Antimicrobial proteins

ABSTRACT

A new family of antimicrobial proteins is described. Prototype proteins can be isolated from  Macadamia integrifolia  as well as other plant species. DNA encoding the protein is also described as well as DNA constructs which can be used to express the antimicrobial protein or to introduce the antimicrobial protein into a plant. Compositions comprising the antimicrobial protein or the antimicrobial protein per se can be administered to plants or mammalian animals to combat microbial infestation.

TECHNICAL FIELD

[0001] This invention relates to isolated proteins which exertinhibitory activity on the growth of fungi and bacteria, which fungi andbacteria include some microbial pathogens of plants and animals. Theinvention also relates to recombinant genes which include sequencesencoding the proteins, the expression products of which recombinantgenes can contribute to plant cells or cells of other organism's defenceagainst invasion by microbial pathogens. The invention further relatesto the use of the proteins and/or genes encoding the proteins for thecontrol of microbes in human and veterinary clinical conditions.

BACKGROUND ART

[0002] Microbial diseases of plants are a significant problem to theagricultural and horticultural industries. Plant diseases in generalcause millions of tonnes of crop losses annually with fungal andbacterial diseases responsible for significant portions of these losses.One possible way of combating fungal and bacterial diseases is toprovide transgenic plants capable of expressing a protein or proteinswhich in some way increase the resistance of the plant to pathogenattack. A simple strategy is to first identify a protein withantimicrobial activity in vitro, to clone or synthesise the DNA sequenceencoding the protein, to make a chimaeric gene construct for efficientexpression of the protein in plants, to transfer this gene to transgenicplants and to assess the effect of the introduced gene on resistance tomicrobial pathogens by comparison with control plants.

[0003] The first and most important step in the strategy for diseasecontrol described above is to identify, characterise and describe aprotein with strong antimicrobial activity. In recent years, manydifferent plant proteins with antimicrobial and/or antifungal activityhave been identified and described. These proteins have been categorisedinto several classes according to either their presumed mode of actionand/or their amino acid sequence homologies. These classes include thefollowing: chitinases (Roberts, W. K. et al. [1986] Biochim. Biophys.Acta 880:161-170); β-1,3-glucanases (Manners, J. D. et al. [1973]Phytochemistry 12:547-553); thionins (Bolmann, H. et al. [1988] EMBO J.7:1559-1565 and Fernadez de Caleya, R. et al. [1972] Appl. Microbiol.23:998-1000); permatins (Roberts, W. K. et al. [1990] J. Gen. Microbiol.136:1771-1778 and Vigers, A. J. et al [1991] Mol. Plant-MicrobeInteract. 4:315-323); ribosome-inactivating proteins (Roberts, W. K. etal. [1986] Biochini. Biophys. Adcta 880:161-170 and Leah, R. et al.[1991] J. Biol. Chem. 266:1564-1573); plant defensins (Terras, F. R. G.et al. [1995] The Plant Cell 7:573-588); chitin binding proteins (DeBolle, M. F. C. et al. [1992] Plant Mol. Biol. 22:1187-1190 and VanParijs, J. et al. [1991] Planta 183:258-264); thaumatin-like, orosmotin-like proteins (Woloshuk, C. P. et al. [1991] The Plant Cell3:619-628 and Hejgaard, J. [1991] FEBS Letts. 291:127-131); PR1-typeproteins (Niderman, T. et al. [1995] Plant Physiol. 108:17-27.) and thenon-specific lipid transfer proteins (Terras, F. R. G. et al. [1992]Plant Physiol. 100:1055-1058 and Molina, A. et al. [1993] FEBS Letts.3166:119-122). Another class of antimicrobial proteins from plants isthe knottin or knottin-like antimicrobial proteins (Cammue, B.P. A. etal. [1992] J. Biol. Chem. 67:2228-2233; Broekaert W. F. et al. (1997)Crit. Rev. in Plant Sci. 16(3):297-323). A class of antimicrobialproteins termed 4-cysteine proteins has also been reported in theliterature which class includes Maize Basic Protein (MBP-1) (Duvick, J.P. et al. [1992] J. Biol. Chem. 267:18114-18120). A novel antimicrobialprotein which does not fit into any previously described class ofantimicrobial proteins has also been isolated from the seeds ofMacadamia integrifolia termed MiAMP1 (Marcus, J. P. et al. [1997] Eur.J. Biochem. 244:743-749). In addition, plants are not the sole source ofantimicrobial proteins and there are many reports of the isolation ofantimicrobial proteins from animal and microbial cells (reviewed inGabay, J. E. [1994] Science 264:373-374 and in “Antimicrobial peptides”[1994] CIBA Foundation Symposium 186, John Wiley and Sons Publ.,Chichester, UK).

[0004] There is evidence that the ectopic expression of genes encodingproteins that have in vitro antimicrobial activity in transgenic plantscan result in increased resistance to microbial pathogens. Examples ofthis engineered resistance include transgenic plants expressing genesencoding: a plant chitinase, either alone (Broglie, K. et al. [1991]Science 254:1194-1197) or in combination with a 1,3-glucanase (Van denElzen, P. J. M. et al. [1993] Phil. Trans. Roy. Soc. 342:271-278); aplant defensin (Terras, F. R. G. et al. [1995] The Plant Cell7:573-588); an osmotin-like protein (Liu, D. et al. [1994] Proc. Natl.Acad. Sci. USA 91:1888-1892); a PR1-class protein (Alexander, D. et al.[1993] Proc. Natl. Acad. Sci. USA 90:7327-7331) and aribosome-inactivating protein (Logemann, J. et al. [1992] Bio/Technology10:305-308).

[0005] Although the potential use of antimicrobial proteins forengineering disease resistance in transgenic plants has been describedextensively, there are other applications which are worthy of mention.Firstly, highly potent antimicrobial proteins can be used for thecontrol of plant disease by direct application (De Bolle, M. F. C. etal. [1993] in Mechanisms of Plant Defence Responses, B. Fritig and M.Legrand eds., Kluwer Acad. Publ., Dordrecht, NL, pp. 433-436). Inaddition, antimicrobial peptides have potential therapeutic applicationsin human and veterinary medicine. Although this has not been describedfor peptides of plant origin it is being actively explored with peptidesfrom animals and has reached clinical trials (Jacob, L. and Zasloff, M.[1994] in “Antimicrobial Peptides”, CIBA Foundation Symposium 186, JohnWiley and Sons Publ., Chichester, UK, pp. 197-223).

[0006] Antimicrobial proteins exhibit a variety of three-dimensionalstructures which will determine in large part the activity which theymanifest. Many of the global structures exhibited by these proteins havebeen determined (Broekaert W.F. et a. (1997) Crit. Rev. in Plant Sci.16(3):297-323). A large factor in determining the stability of theseproteins is the presence of disulfide bridges between various cysteineslocated in a helical and β-sheet regions. Many peptides with toxicactivity such as conotoxin are well known to be stabilized by disulfidebridges (see for example Hill, J. M. et al. (1996) Biochemistry 35(27):8824-8835). In the case of the conotoxin referenced above, a compactstructure is formed consisting of a helix, a small -hairpin, acis-hydroxyproline, and several turns. The molecule is stabilized bythree disulfide bonds, two of which connect the α-helix and the β-sheet,forming a solid structural core. Interestingly, eight arginine andlysine side chains in this molecule project into the solvent in a radialorientation relative to the core of the molecule. These cationic sidechains form potential sites of interaction with anionic sites onpathogen membranes (Hill, J. M. et al. supra).

[0007] The invention described herein constitutes previouslyundiscovered and thus novel proteins with antimicrobial activity. Theseproteins can be isolated from Macadamia integrifolia (Mi) seeds or fromcotton or cocoa seeds. In addition, protein fragments which areantifungal can be derived from larger seed storage proteins containingregions of substantial similarity to the antimicrobial proteins frommacadamia described here. Examples of seed storage proteins whichcontain regions similar to the proteins which have been purified can beseen in FIG. 4. Macadamia integrifolia belongs to the family Proteaceae.M. integrifolia, also known as Bauple Nut or Queensland Nut, isconsidered by some to be the world's best edible nut. Cotton (Gossypiumhirsutum) belongs to the family Malvaceae and is cultivated extensivelyfor its fiber. Cocoa (Threobroma cacao) belongs to the familySterculiaceae and is used around the world for a wide variety of cocoaproducts.

[0008] The fact that both the macadamia and cocoa antimicrobial proteinsare found in edible portions of these plants makes these peptidesattractive for use in genetic engineering for disease resistance sincetrangenic plants expressing these proteins are unlikely to show addedtoxicity. Proteins may also be safe for human and veterinary use.

SUMMARY OF THE INVENTION

[0009] According to a first embodiment of the invention, there isprovided a protein fragment having antimicrobial activity, wherein saidprotein fragment is selected from:

[0010] (i) a polypeptide having an amino acid sequence selected from:

[0011] residues 29 to 73 of SEQ ID NO: 1

[0012] residues 74 to 116 of SEQ ID NO: 1

[0013] residues 117 to 185 of SEQ ID NO: 1

[0014] residues 186 to 248 of SEQ ID NO: 1

[0015] residues 29 to 73 of SEQ ID NO: 3

[0016] residues 74 to 186 of SEQ ID NO: 3

[0017] residues 167 to 185 of SEQ ID NO: 3

[0018] residues 186 to 248 of SEQ ID NO: 3

[0019] residues 1 to 32 of SEQ ID NO: 5

[0020] residues 33 to 75 of SEQ ID NO: 5

[0021] residues 76 to 144 of SEQ ID NO: 5

[0022] residues 145 to 210 of SEQ ID NO: 5

[0023] residues 34 to 80 of SEQ ID NO: 7

[0024] residues 81 to 140 of SEQ ID NO: 7

[0025] residues 33 to 79 of SEQ ID NO: 8

[0026] residues 80 to 11 9 of SEQ ID NO: 8

[0027] residues 120 to 161 of SEQ ID NO: 8

[0028] residues 32 to 91 of SEQ ID NO: 21

[0029] residues 25 to 84 of SEQ ID NO: 22

[0030] residues 29 to 94 of SEQ ID NO: 24

[0031] residues 31 to 85 of SEQ ID NO: 25

[0032] residues 1 to 23 of SEQ ID NO: 26

[0033] residues 1 to 17 of SEQ ID NO: 27

[0034] residues 1 to 28 of SEQ ID NO: 28;

[0035] (ii) a homologue of (i);

[0036] (iii) a polypeptide containing a relative cysteine spacing ofC-2X-C-3X-C-(10-12)X-C-3X-C-3X-C wherein X is any amino acid residue,and C is cysteine;

[0037] (iv) a polypeptide containing a relative cysteine andtyrosine/phenylalanine spacing of Z-2X-C-3X-C-(10-12)X-C-3X-C-3X-Zwherein X is any amino acid residue, and C is cysteine, and Z istyrosine or phenylalanine;

[0038] (v) a polypeptide containing a relative cysteine spacing ofC-3X-C-(10-12)X-C-3X-C wherein X is any amino acid residue, and C iscysteine;

[0039] (vi) a polypeptide with substantially the same spacing ofpositively charged residues relative to the spacing of cysteine residuesas (i); and

[0040] (vii) a fragment of the polypeptide of any one of (i) to (vi)which has substantially the same antimicrobial activity as (i).

[0041] According to a second embodiment of the invention, there isprovided a protein containing at least one polypeptide fragmentaccording to the first embodiment, wherein said polypeptide fragment hasa sequence selected from within a sequence comprising SEQ ID NO: 1, SEQID NO: 3 or SEQ ID NO: 5.

[0042] According to a third embodiment of the invention, there isprovided a protein having a sequence selected from SEQ ID NO: 1, SEQ IDNO: 3 or SEQ ID NO: 5.

[0043] According to a fourth embodiment of the invention, there isprovided an isolated or synthetic DNA encoding a protein according tothe first embodiment According to a fifth embodiment of the invention,there is provided a DNA construct which includes a DNA according to thefourth embodiment operatively linked to elements for the expression ofsaid encoded protein.

[0044] According to a sixth embodiment of the invention, there isprovided a transgenic plant harbouring a DNA construct according to thefifth embodiment.

[0045] According to a seventh embodiment of the invention, there isprovided reproductive material of a transgenic plant according to thesixth embodiment.

[0046] According to an eighth embodiment of the invention, there isprovided a composition comprising an antimicrobial protein according tothe first embodiment together with an agriculturally-acceptable carrierdiluent or excipient.

[0047] According to a ninth embodiment of the invention, there isprovided a composition comprising an antimicrobial protein according tothe first embodiment together with an pharmaceutically-acceptablecarrier diluent or excipient.

[0048] According to a tenth embodiment of the invention, there isprovided a method of controlling microbial infestation of a plant, themethod comprising:

[0049] i) treating said plant with an antimicrobial protein according tothe first embodiment or a composition according to the eighthembodiment; or

[0050] ii) introducing a DNA construct according to the fifth embodimentinto said plant.

[0051] According to an eleventh embodiment of the invention, there isprovided a method of controlling microbial infestation of a mammaliananimal, the method comprising treating the animal with an antimicrobialprotein according to the first embodiment or a composition according tothe ninth embodiment.

[0052] According to a twelfth embodiment of the invention, there isprovided a method of preparing an antimicrobial protein, which methodcomprises the steps of:

[0053] a) obtaining or designing an amino acid sequence which forms ahelix-turn-helix structure;

[0054] b) replacing individual residues to achieve substantially thesame distribution of positively charged residues and cysteine residuesas in one or more of the amino acid sequences shown in FIG. 4;

[0055] c) synthesising a protein comprising said amino acid sequencechemically or by recombinant DNA techniques in liquid culture; and

[0056] d) if necessary, forming disulphide linkages between saidcysteine residues.

[0057] Other embodiments of the invention include methods for producingantimicrobial protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1 shows the results of cation-exchange chromatography of thebasic protein fraction of a Macadamia integrifolia extract with theresults of a bioassay for antimicrobial activity shown for fractions inthe region of MiAMP2c elution.

[0059]FIG. 2 shows the results of including 1 mM Ca²⁺ in a parallelbioassay of fractions from the cation-exchange separation.

[0060]FIG. 3 shows a reverse-phase HPLC profile of highly inhibitoryfractions containing MiAMP2c from the cation-exchange separation inFIGS. 1 and 2 together with % growth inhibition exhibited by the HPLCfractions.

[0061]FIG. 4 shows the amino acid sequences of MiAMP2a, b, c and d andprotein fragments derived from other seed storage proteins which containregions of homology to the MiAMP2 series of antimicrobial proteins.

[0062]FIG. 5 shows an example of a synthetic nucleotide sequence whichcan be used for the expression and secretion of MiAMP2c in transgenicplants.

[0063]FIG. 6 shows the alignment of clones 1-3 from macadamia containingMiAMP2a, b, c and d subunits together with sequences from cocoa andcotton vicilin seed storage proteins which exhibit significant homologyto the macadamia clones.

[0064]FIG. 7 displays a series of secondary structure predictions forMiAMP2c.

[0065]FIG. 8 shows a three-dimensional model of the MiAMP2c protein.

[0066]FIG. 9 shows stained SDS-PAGE gels of protein fractions at variousstages in the expression and purification of TcAMP1 (Theobroma cacaosubunit 1), MiAMP2a, MiAMP2b, MiAMP2c and MiAMP2d expressed in E. coliliquid culture.

[0067]FIG. 10 shows the reverse-phase HPLC purification of cocoa subunit2 (TcAMP2) after the initial purification step using Ni-NTA media.

[0068]FIG. 11 shows a western blot of crude protein extracts fromvarious plant species using rabbit antiserum raised to MiAMP2c.

[0069]FIG. 12 shows a cation-exchange fractionation of the Stenocarpussinuatus basic protein fraction along with the accompanying western blotwhich shows the presence of immunologically-related proteins in a rangeof fractions.

[0070]FIG. 13 shows a reverse-phase HPLC separation of Stenocarpussinuatus cation-exchange fractions which had previously reacted withMiAMP2c antibodies (see FIG. 14). A western blot is also presented whichreveals the presence of putative MiAMP2c homologues in individual HPLCfractions.

[0071]FIG. 14 is a map of the binary vector pPCV91-MiAMP2c as an exampleof a vector that can be used to express these antimicrobial proteins intransgenic plants.

[0072]FIG. 15 shows a western blot to detect MiAMP2c expressed intransgenic tobacco plants.

BEST MODE AND OTHER MODES FOR CARRYING OUT THE INVENTION

[0073] The following abbreviations are used hereafter: EDTAethylenediaminetetraacetic acid IPTG Isopropyl-β-D-thiogalactopyranosideMeCN methyl cyanide (acetonitrile) Mi Macadamia integrifolia MiAMP2Macadamia integrifolia antimicrobial protein series number 2 Ni-NTANickel-nitrilotriacetic acid chromatography media ND not determined PCRpolymerase chain reaction PMSF phenylmethylsulphonyl fluoride SDS-PAGEsodium-dodecylsulphate polyacrylamide gel electrophoresis TFAtrifluoroacetate

[0074] The term homologue is used herein to denote any polypeptidehaving substantial similarity in composition and sequence to thepolypeptide used as the reference. The homologue of a referencepolypeptide will contain key elements such as cysteine or other residuesspaced at identical intervals such that a substantially similarthree-dimensional global structure is adopted by the homologue ascompared to the reference. The homologue will also exhibit substantiallythe same antimicrobial activity as the reference protein.

[0075] The present inventors have identified a new class of proteinswith antimicrobial activity. Prototype proteins can be isolated fromseeds of Macadamia integrifolia. The invention thus providesantimicrobial proteins per se and also DNA sequences encoding theseantimicrobial proteins.

[0076] The invention also provides amino acid sequences of proteinswhich are homologous to the prototype antimicrobial proteins fromMacadamia integrifolia. Thus, in addition to the antimicrobial proteinsfrom Macadamia, this invention also provides amino acid sequences ofhomologues from other species which have hitherto been unrecognized ashaving antimicrobial activity.

[0077] While the first antimicrobial protein in the present series wasisolated directly from Macadamia integrifolia, additional antimicrobialproteins were identified through cloning efforts, homology searches andsubsequent antimicrobial testing of the encoded proteins afterexpression in and purification from liquid culture. After the firstprotein from this series was purified from macadamia and termed MiAMP2,clones were obtained which encoded a preproprotein containing MiAMP2.This large protein (666 amino acids), represented by several almostidentical clones, contained four adjacent regions with significantsimilarity to the purified antimicrobial protein fragment (MiAMP2) whichitself was found to lie within region three in the cloned nucleotidesequence; hence the purified antimicrobial protein is termed MiAMP2c.Other fragments contained in the 666-amino-acid clone are termedMiAMP2a, b and d as per their locations in the cloned nucleotidesequence. Several other sequences with significant homology to theMiAMP2a, b, c, and d protein fragments were then identifed in the Entrezdata base. These homologous sequences were contained within larger seedstorage proteins from cotton and cocoa which sequences had not beenpreviously described as containing antimicrobial protein sequences or asexhibiting antimicrobial activity. Fragments of larger seed storageproteins containing sequences homologous to MiAMP2c were tested and arehere demonstrated to exhibit antimicrobial activity. Thus, the inventorshave established a process for obtaining antimicrobial protein fragmentsfrom larger seed storage proteins. In the light of these findings, it isevident that fragments of other seed storage proteins containingsequences similar to the proteins described will also exhibitantimicrobial activity.

[0078] In particular, the 47-amino-acid TcAMP1 (for Theobroma cacaoantimicrobial protein 1) and the 60-amino-acid TcAMP2 sequences werederived from a cocoa vicilin seed storage protein gene sequence (whichcontains 525 amino acids) (Spencer, M. E. and Hodge R. [1992] Planta186:567-576). These derived fragments were then expressed in liquidculture. Cocoa vicilin fragments thus expressed and subsequentlypurified (Examples 10 and 11), were shown to be antimicrobial (Example15). This is the first report that fragments of the cocoa vicilinprotein possess antimicrobial activity. Pools of sequences containingfragments homologous to the MiAMP2c apparently released from cottonvicilin seed storage protein have been shown to possess antimicrobialactivity (Chung, R. P. T. et al. [1997] Plant Science 127:1-16). Thisfinding is clearly embodied in sequences disclosed in this application.

[0079] In addition to showing that cocoa-vicilin-derived fragmentsexhibit antimicrobial activity, there is herein described additionalproteins which exhibit antimicrobial activity. For example, there isdescribed below proteins from Stenocarpus sinuatus which are of similarsize to MiAMP2 subunits, react with MiAMP2c antiserum, and containsequences homologous to MiAMP2 proteins (see FIG. 4). Based on theevidence provided herein, sequences homologous to the MiAMP2c subunit(i.e., MiAMP2a, b, d; TcAMP1; TcAMP2; and cotton fragments 1, 2 and3—see FIG. 4) constitute proteins which contain the fragment withantimicrobial activity. The antimicrobial activity of MiAMP2 fragmentsfrom macadamia, and the TcAMP1 and 2 fragments from cocoa, isexemplified below. R. P. T. Chung et al. (Plant Science 127:1-16 [1997])have demonstrated that the cotton fragments exhibit antimicrobialactivity. Other antimicrobial proteins can also be derived from seedstorage proteins such as peanut allergen Ara h (Burks, A. W. et al.[1995] J. Clin. Invest. 96 (4), 1715-1721), maize globulin (Belanger, F.C. and Kriz, A. L.[1991] Genetics 129 (3), 863-872), barley globulin(Heck, G. R. et al. [1993] Mol. Gen. Genet. 239 (1-2), 209-218), andsoybean. conglycinin (Sebastiani, F. L. et al. [1990] Plant Mol. Biol.15 (1), 197-201), all of which contain the same key elements which arepresent in the sequences which are here shown to exhibit antimicrobialactivity.

[0080] The proteins which contain regions of sequence homologous toMiAMP2 (as in FIG. 4) can be used to construct nucleotide sequencesencoding 1) the active fragments of larger proteins, or 2) fusions ofmultiple antimicrobial fragments. This can be done using standard codontables and cloning methods as described in laboratory manuals such asCurrent Protocols in Molecular Biology (copyright 1987-1995 edited byAusubel F. M. et al. and published by John Wiley & Sons, Inc., printedin the USA). Subsequently, these can be expressed in liquid culture forpurification and testing, or the sequences can be expressed intransgenic plants after placing them in appropriate expression vectors.

[0081] The antimicrobial proteins per se will manifest a particularthree-dimensional structure which may be determined using X-raycrystallography or nuclear magnetic resonance techniques. This structurewill be responsible in large part for the antimicrobial activity of theprotein. The sequence of the protein can also be subjected to structureprediction algorithms to assess whether any secondary structure elementsare likely to be exhibited by the protein (see Example 8 and FIG. 7).Secondary structures, thus predicted, can then be used to modelthree-dimensional global structures. Although three-dimensionalstructure prediction is not feasible for most proteins, the secondarystructure predictions for MiAMP2c were sufficiently simple and clearthat a three-dimensional model structure has been obtained for theMiAMP2c protein. Homologues exhibiting the same cysteine spacing andother key elements will also adopt the same three-dimensional structure.Example 8 shows that the structure most likely to be adopted by MiAMP2c(and homologues) is a helix-turn-helix structure stabilised by at leasttwo disulfide bridges connecting the two antiparallel α-helical segments(see FIG. 8). Additional stabilisation can be provided by an extradisulfide bridge (e.g., as in MiAMP2b) or by a hydrophobic ring-stackinginteraction between tyrosine and/or phenylalanine residues (e.g.,MiAMP2a and MiAMP2c), each located on the same face of the α-helicalsegments as the normally present cysteine residues which participate inthe 2 disulfide linkages mentioned above. NMR signals exhibited byMiAMP2c are consistent with the three-dimensional global model producedfrom the secondary-structure predictions mentioned above.

[0082] It will be appreciated that one skilled in the art could take aprotein with known structure, alter the sequence significantly, and yetretain the overall three-dimensional shape and antimicrobial activity ofthe protein. One aspect of the structure that most likely could not bealtered without seriously affecting the structure (and, therefore, theactivity of the protein) is the content and spacing of the cysteineresidues since this would disrupt the formation of disulfide bonds whichare critical to a) maintaining the overall structure of the proteinand/or b) making the protein more resistant to denaturation andproteolysis (stabilizing the protein structure). In particular, it isessential that cysteine residues reside on one face of the helix inwhich they are contained. This can best be accomplished by maintaining athree-residue spacing between the cysteine residues within each helix,but, can also be accomplished with a two-residue interval between thecysteine residues—provided the cysteines on the other helical segmentare separated by three residues (i.e.,C-X-X-C-X-X-X-C-nX-C-X-X-X-C-X-X-X-C where C is cysteine, X is any aminoacid, and n is the number of residues forming a turn between the twoα-helical segments). Aromatic tyrosine (or phenylalanine) residues canalso function to add stability to the protein structure if they arelocated on the same face of the helix as the cysteine side chains. Thiscan be accomplished by providing appropriate spacing of two or threeresidues between the aromatic residue and the proximate cysteine residue(i.e., Z-X-X-C-X-X-X-C-nX-C-X-X-X-C-X-X-X-Z where Z is tyrosine orphenylalanine).

[0083] The distribution of positive (and negative) charges on thevarious surfaces of the protein will also serve a critical role indetermining the structure and activity of the protein. In particular,the distribution of positively-charged residues in an α-helical regionof a protein can result in positive charges lying on one face of thehelix or may result in the charged residues being concentrated in someparticular portion of the molecule. An alternative distribution ofpositively charged residues is for them to project into the solvent in aradial orientation to the core of the protein. This orientation ispredicted for several of the MiAMP2 homologues (data not shown). Thespacing which is required for positioning of the residues on one face ofthe helix or the-spacing required to accomplish a radial orientationfrom the core can easily be determined by one skilled in the art using ahelical wheel plot with the sequence of interest. A helical wheel plotuses the fact that, in α-helices, each turn of the helix is composed of3.6 residues on average. This number translates to 100° of rotationaltranslation per residue making it possible to construct a plot showingthe distribution of side chains in a helical region. FIG. 8 shows howthe spacing of charged residues can lead to most of the positivelycharged side chains being localised on one face of the helix. It will beappreciated by one of skill in the art that positive charges areconferred by arginine and lysine residues.

[0084] In order for the protein to develop into a helix-turn-helixstructure, it is also necessary to have particular residues that favorα-helix formation and that also favor a turn structure in the middleportion of the amino acid sequence (and disfavor a helical structure inthe turn region). This can be accomplished by a proline residue orresidues in the middle of the turn segment as seen with many of theMiAMP2 homologues. When proline is not present, glycine can alsocontribute to breaking a continuous helix structure, and inducing theformation of a turn at this position. In one case (i.e., TcAMP1), itappears that serine may be taking on this role. It will be appreciatedthat the residues in this region of the protein will usually favor theformation of a turn structure; residues which fulfill this requirementinclude proline, glycine, serine, and aspartic acid; but, other residuesare also allowed.

[0085] The DNA sequences reported here are an extremely powerful toolwhich can be used to obtain homologous genes from other species. Usingthe DNA sequences, one skilled in the art can design and synthesiseoligonucleotide probes which can be used to screen cDNA libraries fromother species of plants for the presence of genes encoding antimicrobialproteins homologous to the ones described here. This would simplyinvolve construction of a cDNA library and subsequent screening of thelibrary using as the oligonucleotide probe one or part of one of thesequences reported here (such as sequence ID. No. 2 or the PCR fragmentdescribed in Example 9). Other oligonucleotide sequences coding forproteins homologous to MiAMP2 can also be used for this purpose (e.g.,DNA sequences corresponding to cotton and cocoa vicilins). Making andscreening of a cDNA library can be carried out by purchasing a kit forsaid purpose (e.g., from Stratagene) or by following well establishedprotocols described in available DNA cloning manuals (see CurrentProtocols in Molecular Biology, supra). It is relatively straightforward to construct libraries of various species and to specificallyisolate vicilin homologues which are similar to the Macadamia, cotton,or cocoa vicilins by using a simple DNA hybridization technique toscreen such libraries. Once cloned, these vicilin-related sequences canthen be examined for the presence of MiAMP2-like subunits. Such subunitscan easily be expressed in E. coli using the system described inExamples 10 and 11. Subsequently, these proteins can also be expressedin transgenic.

[0086] Genes, or fragments thereof, under the control of a constitutiveor inducible promoter, can then be cloned into a biological system whichallows expression of the protein encoded thereby. Transformation methodsallowing for the protein to be expressed in a variety of systems areknown. The protein can thus be expressed in any suitable system for thepurpose of producing the protein for further use. Suitable hosts for theexpression of the protein include E. coli, fungal cells, insect cells,mammalian cells, and plants. Standard methods for expressing proteins insuch hosts are described in a variety of texts including section 16(Protein Expression) of Current Protocols in Molecular Biology (supra).

[0087] Plant cells can be transformed with DNA constructs of theinvention according to a variety of known methods (Agrobacterium, Tiplasmids, electroporation, micro-injections, micro-projectile gun, andthe like). DNA sequences encoding the Macadamia integrifoliaantimicrobial protein subunits (i.e. fragments a, b, c, or d from theMiAMP2 clones) as well as DNA coding for other homologues can be used inconjunction with a DNA sequence encoding a preprotein from which themature protein is produced. This preprotein can contain a native orsynthetic signal peptide sequence which will target the protein to aparticular cell compartment (e.g., the apoplast or the vacuole). Thesecoding sequences can be ligated to a plant promoter sequence that willensure strong expression in plant cells. This promoter sequence mightensure strong constitutive expression of the protein in most or allplant cells, it may be a promoter which ensures expression in specifictissues or cells that are susceptible to microbial infection and it mayalso be a promoter which ensures strong induction of expression duringthe infection process. These types of gene cassettes will also include atranscription termination and polyadenylation sequence 3′ of theantimicrobial protein coding region to ensure efficient production andstabilisation of the mRNA encoding the antimicrobial proteins. It ispossible that efficient expression of the antimicrobial proteinsdisclosed herein might be facilitated by inclusion of their individualDNA sequences into a sequence encoding a much larger protein which isprocessed in planta to produce one or more active MiAMP2-like fragments.

[0088] Gene cassettes encoding the MiAMP2 series antimicrobial proteins(i.e., MiAMP2a, b, c, or d; or all of the subunits together; or theentire MiAMP2 clone) or homologues of the MiAMP2 proteins as describedabove can then be expressed in plant cells using two common methods.Firstly, the gene cassettes can be ligated into binary vectors carrying:i) left and right border sequences that flank the T-DNA of theAgrobacterium tumefaciens Ti plasmid; ii) a suitable selectable markergene for the selection of antibiotic resistant plant cells; iii) originsof replication that function in either A. tumefaciens or Escherichiacoli; and iv) antibiotic resistance genes that allow selection ofplasmid-carrying cells of A. tumefaciens and E. coli. This binary vectorcarrying the chimaeric MiAMP2 encoding gene can be introduced by eitherelectroporation or triparental mating into A. tumefaciens strainscarrying disarmed Ti plasmids such as strains LBA4404, GV3101, and AGL1or into A. rhizogenes strains such as A4 or NCCP1885. TheseAgrobacterium strains can then be co-cultivated with suitable plantexplants or intact plant tissue and the transformed plant cells and/orregenerants selected using antibiotic resistance.

[0089] A second method of gene transfer to plants can be achieved bydirect insertion of the gene in target plant cells. For example, anMiAMP2-encoding gene cassette can be co-precipitated onto gold ortungsten particles along with a plasmid encoding a chimaeric gene forantibiotic resistance in plants. The tungsten particles can beaccelerated using a fast flow of helium gas and the particles allowed tobombard a suitable plant tissue. This can be an embryogenic cellculture, a plant explant, a callus tissue or cell suspension or anintact meristem. Plants can be recovered using the antibiotic resistancegene for selection and antibodies used to detect plant cells expressingthe MiAMP2 proteins or related fragments.

[0090] The expression of MiAMP2 proteins in the transgenic plants can bedetected using either antibodies raised to the protein(s) or usingantimicrobial bioassays. These and other related methods for theexpression of MiAMP2 proteins or fragments thereof in plants aredescribed in Plant Molecular Biology (2nd ed., edited by Gelvin, S. B.and Schilperoort, R. A., ©1994, published by Kluwer Academic Publishers,Dordrecht, The Netherlands)

[0091] Both monocotyledonous and dicotyledonous plants can betransformed and regenerated. Examples of genetically modified plantsinclude maize, banana, peanut, field peas, sunflower, tomato, canola,tobacco, wheat, barley, oats, potato, soybeans, cotton, carnations,roses, sorghum. These, as well as other agricultural plants can betransformed with the antimicrobial genes such that they would exhibit agreater degree of resistance to pathogen attack. Alternatively, theproteins can be used for the control of diseases by topologicalapplication.

[0092] The invention also relates to application of antimicrobialprotein in the control of pathogens of mammals, including humans. Theprotein can be used either in topological or intravenous applicationsfor the control of microbial infections.

[0093] As indicated above in the description of the tenth embodiment,the invention includes within its scope the preparation of antimicrobialproteins based on the prototype MiAMP2 series of proteins. New sequencescan be designed from the MiAMP2 amino acid sequences which substantiallyretain the distribution of positively charged residues relative tocysteine residues as found in the MiAMP2 proteins. The new sequence canbe synthesised or expressed from a gene encoding the sequence in anappropriate host cell. Suitable methods for such procedures have beendescribed above. Expression of the new protein in a geneticallyengineered cell will typically result in a product having a correctthree-dimensional structure, including correctly formed disulphidelinkages between cysteine residues. However, even if the protein ischemically synthesised, methods are known in the art for furtherprocessing of the protein to break undesireable disulfide bridges andform the bridges between the desired cysteine residues to give thedesired three-dimensional structure should this be necessary.

[0094]Macadamia integrifolia antimicrobial proteins series number 2

[0095] As indicated above, a new series of potent antimicrobial proteinshas been identified in the seeds of Macadamia integrifolia. The proteinscollectivelly are called the MiAMP2 series of antimicrobial proteins (orMiAMP2 proteins) because they are all found on one large preproproteinwhich is processed into smaller subunits, each exhibiting antimicrobialactivity; they represent the second class of antimicrobial proteinsisolated from Macadamia integrifolia. Each protein fragment of theseries has a characteristic pI value. MiAMP2a, b, c, and d subunits asshown in FIG. 4 have predicted pI values of 4.4, 4.6, 11.5, and 11.6respectively (predicted using raw sequence data without the His tag orcleavage sequences associated with expression of fragments in the vectorpET16b), and contain two sets of CXXXC motifs which are important instabilising the three-dimensional structure of the protein through theformation of disulfide bonds. Additionally, the proteins contain eitheran added set of aromatic (tyrosine/phenylalanine) residues or an addedset of cysteine residues located at positions which would give morestability to the helix-turn-helix structure as described above and inExample 8.

[0096] The amino acid sequences of the MiAMP2 series of proteins sharesignificant homology with fragments of previously described proteins insequence databases (Swiss Prot and Non-redundant databases) searchedusing the BLASTP algorithm (Altschul, S. F. et al. [1990] J. Mol. Biol.215:403). In particular, MiAMP2a, b, c and d sequences exhibitsignificant similarity with regions of cocoa vicilin and cotton vicilin(as seen in FIG. 6). Some similarity is also seen with fragments fromother seed storage proteins of peanut (Burks, A. W. et al. [1995] J.Clin. Invest. 96 (4), 1715-1721), maize (Belanger, F. C. and Kriz, A.L.[1991] Genetics 129 (3), 863-872), barley (Heck, G. R. et al. [1993]Mol. Gen. Genet. 239 (1-2), 209-218), and soybean (Sebastiani, F. L. etal. [1990] Plant Mol. Biol. 15 (1), 197-201). Although, in some casesthe homology is not extremely high (for example, 18% identity betweenMiAMP2a and cotton subunit 1; see FIG. 4), the spacing of the main fourcysteine residues is conserved in all subunits and homologues. Inaddition, both cotton and cocoa vicilin-derived subunits retain theconserved tyrosine or phenylalanine residues as additional stabilisersof the tertiary structure. The cotton and cocoa vicilins with 525 and590 amino acids, respectively, are much larger proteins than MiAMP2c (47amino acids) (see FIGS. 4 and 6). Although MiAMP2 subunits also sharesome homology with MBP-1 antimicrobial protein from maize (Duvick, J. P.et al. (1992) J Biol Chem 267:18814-20) the number of residues betweenthe CXXXC motifs is 13 which puts MBP-1 outside the specifications forthe spacing given here in this application. MBP-1 is also a smallerprotein (33 amino acids), overall, than the sequences claimed here andthere is no evidence available the MBP-1 is derived from a larger seedstorage protein other than some similarity with a portion of miazeglobulin protein. However, MBP-1 cannot be derived from from the maizeglobulin since maize globulin contains 10 residues between the two CXXXCmotifs while MBP-1 contains 13. The alignments in FIGS. 4 and 6 show thesimilarity in cysteine spacing between MiAMP2 subunits and the cocoa andcotton vicilin-derived molecules. The cysteine and the aromatictyrosine/phenylalanine residues in FIGS. 4 and 6 are highlighted withbold underlined text. FIG. 4 also shows the alignment of additionalproteins which can be expressed in liquid culture and shown to exhibitantimicrobial activity.

[0097] All of the MiAMP2 homologues that have been tested exhibitantifungal activity. MiAMP2 homologues show very significant inhibitionof fungal growth at concentrations as low as 2 μg/ml for some of thepathogens/microbes against which the proteins were tested. Thus they canbe used to provide protection against several plant diseases. MiAMP2homologues can be used as fungicides or antibiotics by application toplant parts. The proteins can also be used to inhibit growth ofpathogens by expressing them in transgenic plants. The proteins can alsobe used for the control of human pathogens by topological application orintravenous injection. One characteristic of the proteins is thatinhibition of some microbes is suppressed by the presence of Ca²⁺ (1mM). An example of this effect is provided for MiAMP2c subunit in Table1.

[0098] Some of the MiAMP2 proteins and homologues could also function asinsect control agents. Since some of the proteins are extremely basic(e.g., pI>11.5 for MiAMP2c and d subunits), they would maintain a strongnet-positive charge even in the highly alkaline environment of an insectgut. This strong net-positive charge would enable it to interact withnegatively charged structures within the gut. This interaction may leadto inefficient feeding, slowing of growth, and possibly death of theinsect pest.

[0099] Non-limiting examples of the invention follow.

EXAMPLE 1 Extraction of Basic Protein from Macadamia integrifolia Seeds

[0100] Twenty five kilograms of Mi nuts (purchased from the MacadamiaNut Factory, Queensland, Australia) were ground in a food processor (TheBig Oscar, Sunbeam) and the resulting meal was extracted for 2-4 hoursat 4° C. with 50 L of an ice-cold extraction buffer containing 10 mMNaH₂PO₄, 15 mM Na₂HPO₄, 100 mM KCl, 2 mM EDTA, 0.75%polyvinylpolypyrolidone, and 0.5 mM phenylmethylsulfonyl fluoride(PMSF). The resulting homogenate was run through a kitchen strainer toremove larger particulate material and then further clarified bycentrifugation (4000 rpm for 15 min) in a large capacity centrifuge.Solid ammonium sulphate was added to the supernatant to obtain 30%relative saturation and the precipitate allowed to form overnight withstirring at 4° C. Following centrifugation at 4000 rpm for 30 min, thesupernatant was taken and ammonium sulphate added to achieve 70%relative saturation. The solution was allowed to precipitate overnightand then centrifuged at 4000 rpm for 30 min in order to collect theprecipitated protein fraction. The precipitated protein was resuspendedin a minimal volume of extraction buffer and centrifuged once again(13,000 rpm×30 min) to remove the any insoluble material yet remaining.After dialysis (10 mM ethanolamine pH 9.0, 2 mM EDTA and 1 mM PMSF) toremove residual ammonium sulphate, the protein solution was passedthrough a Q-Sepharose Fast Plow column (5×12 cm) previously equilibratedwith 10 mM ethanolamine (pH 9), 2 mM in EDTA). The collected flowthroughfrom this column represents the basic (pI>9) protein fraction of theseeds. This fraction was further purified as described in Example 3.

EXAMPLE 2 Antifungal and Antibacterial Activity Assays

[0101] In general, bioassays to assess antifungal and antibacterialactivity were carried out in 96-well microtitre plates. Typically, thetest organism was suspended in a synthetic growth medium consisting ofK₂HPO₄ (2.5 mM), MgSO₄ (50 μM), CaCl₂ (50 μM), FeSO₄ (5 μM), CoCl₂ (0.1μM), CuSO₄ (0.1 μM), Na₂MoO₄ (2 μM), H₃BO₃ (0.5 μM), KI (0.1 μM), ZnSO₄(0.5 μM), MnSO₄ (0.1 μM), glucose (10 g/L), asparagine (1 g/L),methionine (20 mg/L), myo-inositol (2 mg/L), biotin (0.2 mg/L),thiamine-HCl (1 mg/L) and pyridoxine-HCl (0.2 mg/L). The test organismconsisted of bacterial cells, fungal spores (50,000 spores/ml) or fungalmycelial fragments (produced by blending a hyphal mass from a culture ofthe fungus to be tested and then filtering through a fine mesh to removelarger hyphal masses). Fifty microlitres of the test organism suspendedin medium was placed into each well of the microtitre plate. A further50 μl of the test antimicrobial solution was added to appropriate wells.To deal with well-to-well variability in the bioassay, 4 replicates ofeach test solution were done. Sixteen wells from each 96-well plate wereused as controls for comparison with the test solutions.

[0102] Unless otherwise stated, incubation was at 25° C. for 48 hours.All fungi including yeast were grown at 25° C. E. coli were grown at 37°C. and other bacteria were bioassayed at 28° C. Percent growthinhibition was measured by following the absorbance at 600 nm of growingcultures over various time intervals and is defined as 100 times theratio of the average change in absorbance in the control wells minus thechange in absorbance in the test well divided by the average change inabsorbance at 600 nm for the control wells (i.e., [(avg change incontrol wells−change in test well)/(avg change in control wells)]×100).Typically, measurements were taken at 24 hour intervals and the periodfrom 24-48 hours was used for %Inhibition measurements.

EXAMPLE 3 Purification of Antimicrobial Protein from Macadamiaintegrifolia Basic Protein Fraction

[0103] The starting material for the isolation of the Mi antimicrobialprotein was the basic fraction extracted from the mature seeds asdescribed above in Example 1. This protein was further purified bycation exchange chromatography as shown in FIG. 1.

[0104] About 4 g of the basic protein fraction dissolved in 20 mM sodiumsuccinate (pH 4) was applied to an S-Sepharose High Performance column(5×60 cm) (Pharmacia) previously equilibrated with the succinate buffer.The column was eluted at 17 ml/min with a linear gradient of 20 L from 0to 2 M NaCl in 20 mM sodium succinate (pH 4). The eluate was monitoredfor protein by on-line measurement of the absorbance at 280 nm andcollected in 200 ml fractions. Portions of each fraction weresubsequently tested in the antifungal activity assay against Phytopthoracryptogea at a concentration of 100 μg/ml in the presence and absence of1 mM Ca²⁺. Results of bioassays are included in FIGS. 1a and 1 b wherethe elution gradient is shown as a solid line and the shaded barsrepresent %Inhibition. The FIG. 1a assays were conducted without addedCa²⁺ while 1 mM Ca²⁺ was included in the FIG. 1b assays. Fractionationyielded a number of unresolved peaks eluting between 0.05 and 2 M NaCl.A peak eluting at about 16 hours into the separation (fractions 90-92)showed significant antimicrobial activity.

[0105] Fractions showing significant antimicrobial activity were furtherpurified by reversed-phase chromatography. Aliquots of fractions 90-92were loaded onto a Pep-S (C2/C₁₈), column (25×0.93 cm) (Pharmacia)equilibrated with 95% H₂O/5% MeCN/0.1% TFA (=100%A). The column waseluted at 3 ml/min with a 240 ml linear gradient (80 min) from 100%A to100%B (=5% H₂O/95% MeCN/0.1% TFA). Individual peaks were collected,vacuum dried three times in order to remove traces of TFA, andsubsequently resuspended in 500 microlitres of milli-Q water (MilliporeCorporation water purification system) for use in bioassays as describedin Example 2. FIG. 2 shows the HPLC profile of purified fraction 92 fromthe cation-exchange separation shown in FIGS. 1 and 2. Protein elutionwas monitored at 214 nm. The acetonitrile gradient is shown by thestraight line. Individual peaks were bioassayed for antimicrobialactivity: the bars in FIG. 3 show the inhibition corresponding to 15μg/ml of material from each of the fractions. The active protein elutesat approximately 27 min (˜30% MeCN/0.1%TFA) and is called MiAMP2c.

EXAMPLE 4 Purity of Isolated MiAMP2c

[0106] The purity of the isolated antimicrobial protein was verified bynative SDS-PAGE followed by staining with coomassie blue proteinstaining solution. Electrophoresis was performed on a 10-20% tricinegradient gel (Novex) as per the manufacturers recommendations (100 V,1-2 hour separation time). Under these conditions the purified MiAMP2cmigrates as a single discrete band (<10 kDa in size). The detection of asingle major band in the SDS-PAGE analysis together with single peakseluting in the cation-exchange and reversed-phase separations (notshown), gives strong indication that the MiAMP2c preparation is greaterthan 95% pure and therefore the activity of the preparation was almostcertainly due to the MiAMP2c alone and not to a minor contaminatingcomponent. A clean signal in mass spectrometric analysis (Example 5below) also supports this conclusion.

EXAMPLE 5 Mass Spectroscopic Analysis of MiAMP2c

[0107] Purified MiAMP2c was submitted for mass spectroscopic analysis.Approximately 1 μg of protein in solution was used for testing. Analysisshowed the protein to have a molecular weight of 6216.8 Da ±2 Da.Additionally, the protein was subjected to reduction of disulfide bondswith dithiothreitol and alkylation with 4-vinylpyridine. The product ofthis reductionlalkylation was then submitted for mass spectroscopicanalysis and was shown to have gained 427 mass units (i.e. molecularweight was increased by approximately 4×10⁶ Da). The gain in massindicated that four 4-vinylpyridine groups had reacted with the reducedprotein, demonstrating that the protein contains a total of 4 cysteineresidues. The cysteine content has also been subsequently confirmedthrough amino acid sequencing.

EXAMPLE 6 Amino Acid Sequence of MiAMP2c Protein

[0108] Approximately 1 μg of the pure protein which had been reduced andalkylated was subjected to Automated Edman degradation N-terminalsequencing. In the first sequencing run, the sequence of the first 39residues was determined. Subsequently, approximately 1 mg of MiAMP2c wasreacted with Cyanogen Bromide which cleaved the protein on theC-terminal side of Methionine-26. The C-terminal fragment generated bythe cleavage reaction was purified by reversed-phase HPLC and sequenced,yielding the remaining sequence of MiAMP2c (i.e. residues 27-47). Thefull amino acid sequence is RQRDP QQQYE QCQER CQRHE TEPRH MQTCQ QRCERRYEKE KRKQQ KR and represents amino acids 118 to 164 of clone 3 fromExample 9 (see FIG. 6 and SEQUENCE ID NO: 5). In the figure, cysteineresidues are in bold type and underlined to facilitate recognition ofthe spacing patterns. Depending on the number of disulfide bonds thatare formed, the protein mass will range from 6215.6 to 6219.6 Da. Thisis in close agreement with the mass of 6216.8 i 2 Da obtained by massspectrometric analysis (Example 5). The measured mass closelyapproximates the predicted mass of MiAMP2c in a two-disulfide form as isexpected to be the case.

EXAMPLE 7 Synthetic DNA Sequence Coding for MiAMP2c with a LeaderPeptide

[0109] Using standard codon tables it is possible to reverse-translatethe protein sequences to obtain DNA sequences that will code for theantimicrobial proteins. The software program Mac Vector 4.5.3 was usedto enter the protein sequence and obtain a degenerate nucleotidesequence. A codon usage table for tobacco was referenced in order topick codons that would be adequately represented in tobacco for purposesof obtaining high expression in this test plant. A 30 amino-acid leaderpeptide was also designed to ensure efficient processing of the signalpeptide and secretion of the peptide extracellularly. For this purpose,the method of Von Hiejne was used to evaluate a series of possibleleader sequences for probability of cleavage at the correct position[Von Hiejne, G. (1986) Nucleic Acids Research 14(11): 4683-4690]. Inparticular, the amino acid sequence MAWFH VSVCN AVFVV IIIIM LLMFV PVVRG(Sequence ID. No. 11) was found to give an optimal probability ofcorrect processing of the signal peptide immediately following the G(Gly) of this leader sequence. A 5′ untranslated region from tobaccomosaic virus was also added to this synthetic gene to promote highertranslational efficiency [Dowson, M. J., et al. (1994) Plant Mol. Biol.Rep. 12(4):347-357]. The synthetic gene also contains restriction sitesat the 5′ and 3′ ends and immediately 5′ of the start ATG for efficientcloning and subcloning procedures. FIG. 5 shows a synthetic DNA sequencesuitable for use in plant expression experiments. In this Figure, thearrow shows where translation is initiated and the triangular symbolindicates the point of cleavage of the signal peptide.

EXAMPLE 8 Structure Prediction of MiAMP2c Protein

[0110] Using sequence analysis algorithms, putative secondary structuremotifs can be assigned to the protein. Five different algorithms wereused to predict whether α-helices, β-sheets, or turns can occur in theMiAMP2c protein (FIG. 4). Methods were obtained from the followingsources: DPM method, Deleage, G., and Roux, B. (1987) Prot. Eng.1:289-294; SOPMA method, Geourjon, C., and Deleage, G. (1994) Prot. Eng.7:157-164; Gibrat method, Gibrat, J. F., Garnier, J., and Robson, B.(1987) J. Mol. Biol. 198:425-443; Levin method, Levin, J. M., Robson,B., and Garnier, J. (1986) FEBS Lett. 205:303-308; and PhD method, Rost,B., And Sander, C. (1994) Proteins 19:55-72. FIG. 7 shows the predictedlocations of α-helices, β-sheets and turns. The following symbols havebeen used in FIG. 7: C, coil (unstructured); H, alpha helix; E, β-sheet;and S, turn. Underlined residues are those which were predicted toexhibit an α-helical structure by at least 2 separate structureprediction methods; these are represented as helices in FIG. 8.

[0111] It is clear from the secondary structure predictions that theprotein is highly α-helical. While secondary structure prediction isoften difficult and inaccurate, this particular prediction gives a clearindication of the structure of the protein. Examination of thesecondary-structure predictions show a clear preponderance of twoα-helical regions broken by a stretch of about 5-8 residues. This ishighly suggestive of a helix-turn-helix motif.

[0112] Helical wheel analysis of the MiAMP2c amino acid sequence showsthat cysteine residues with a CXXXC spacing will be aligned on one faceof the helix in which they are located Since the cysteines are involvedin disulfide bond formation, the cysteine side chains in one helix mustform covalent bonds with the cysteine side chains located on the otherhelical segment. When the helical segments are arranged in such a way asto bring the cysteine side chains from each respective helix intoproximity with the other cysteine side chains, the resultingthree-dimensional structure is shown in FIG. 8. This structure exhibitsa remarkable distribution of positively charge residues on one face ofthe protein comprised of two helices held together by two disulfidebonds. FIG. 8 shows how the spacing of positively charged residues inhelical regions of this molecule will cause these side chains to lie onone face of the helix. The positively charged residues are the dark sidechains outlined in black. Other dark side chains represent acidicresidues. A proline residue (grey colour marked with a ‘P’) is locatedat the extreme left end of the molecule in the turn region. Solid blacklines show where disulfide bonds connect the two helices. The dottedline shows where the two aromatic hydrophobic residues interact to addstability to the helix-turn-helix structure.

[0113] This helix-turn-helix structure will be adopted by all MiAMP2homologues containing the same cysteine spacing and residues with helixand turn-forming propensities. Other MiAMP2 fragment sequences can besuperimposed onto the global structure shown in FIG. 8. The overallstructure will remain essentially the same but the charge distributionwill vary according to the sequences involved. In the case of MiAMP2b,the dotted line would represent an added disulfide bridge instead of ahydrophobic interaction.

EXAMPLE 9 cDNA Cloning of Genes Corresponding to MiAMP2c

[0114] PCR Amplification of a genomic fragment of the MiAMP2c Gene

[0115] Using the reverse-translated nucleotide sequences, degenerateprimers were made for use in PCR reactions with genomic DNA fromMacadamia. Primer JPM17 sequence was 5′ CAG CAG CAG TAT GAG CAG TG 3′and primer JPM20 degenerate sequence was 5′ TTT TTC GTA (T/T)C(T/G)(G/T)C(T/G) TTC GCA 3′ (SEQ ID NOS: 12 and 13). Primers JPM17 and JPM20were used in PCR amplifications carried out for 30 cycles with 30 sec at95° C., 1 min at 50° C., and 1 min at 72° C. PCR products with sizesclose to those which were expected were directly sequenced (ABI PRISMDye Terminator Cycle Sequencing Ready Reaction Kit from Perkin ElmerCorporation) after excising DNA bands from agarose gels and purifyingthem using a Qiagen DNA clean-up kit. Using this approach, it waspossible to amplify a fragment of DNA of approximately 100 bp. Directsequencing of this nucleotide fragment yielded the nucleotide sequencecorresponding to a portion of the amino acid sequence of theantimicrobial protein MiAMP2c (amino acids 7-39 of FIG. 4). The partialnucleotide sequence obtained from the above-mentioned fragment excludingthe primer sequences was 5′ TCA GAA GCG CTG CCA ACG GCG CGA GAC AGA GCCACG ACA CAT GCA AAT TTG TCA ACA ACG C 3′ (corresponding to base pairs264 to 324 in SEQ ID NO: 6). This sequence can be used for a variety ofpurposes including screening of cDNA and genomic libraries for clones ofMiAMP2 homologues or design of specific primers for PCR amplificationreactions.

[0116] Messenger RNA Isolation from Macadamia Nut Kernels

[0117] Fifty-eight grams of Macadamia nut kernels were ground to powderunder liquid nitrogen using a mortar and pestel. RNA from groundmaterial was then purified using a Guanidine thiocyanate/Cesium chloridetechnique (Current Protocols in Molecular Biology, supra). Using thismethod approximately 5 mg of total RNA was isolated. Messenger RNA wasthen purified from total RNA using a spun column mRNA purification kit(Pharmacia).

[0118] cDNA Library Construction

[0119] A cDNA library was constructed in a lambda ZAP vector using alibrary kit from Stratagene. A total of 6 reactions were performed using25 micrograms of messenger RNA. First and second strand cDNA synthesiswas performed using MMLV Reverse transcriptase and DNA Polymerase I,respectively. After blunting the cDNA with Pfu DNA Polymerase, Eco RIlinker adapters were ligated to the DNA. DNA was then kinased using T4polynucleotide kinase and the DNA subsequently digested with Xho Irestriction endonuclease. At this point cDNA material was fractionatedaccording to size using a sephacryl-S500 column supplied with the kit.DNA was then ligated into the lambda ZAP vector. The vector containingligated insert was then packaged into lambda phage (Gigapack IIIpackaging extract from Stratagene).

[0120] Screening of Library

[0121] The library constructed above was then plated and screened inXL1-blue E. coli bacterial lawns growing in top agarose. Plaquescontaining individual clones were isolated by lifting onto Hybond N+membranes (Amersham LIFE SCIENCE), hybridizing to a radiolabeled versionof the genomic DNA fragment amplified above, imaging of the blot, andpicking of possitive clones for the next round of screening. Aftersecondary and tertiary screening, plaques were sufficiently isolated toallow picking of single clones. Several clones were obtained, andsubsequently the pBK-CMV vector portion from the larger lambda vectorwas excised.

[0122] Sequence of MiAMP2c cDNA Clones

[0123] Vectors (pBK-CMV) containing putative MiAMP2c clones weresequenced to obtain the DNA sequence of the cloned inserts. Seven cloneswere partially sequenced and an additional three clones were fullysequenced (see SEQ ID NOS: 2, 4 and 6 for DNA sequences of the macadamiaclones). Translation of the DNA sequences showed that the full lengthclones encoded highly similar proteins of 666 amino acids. FIG. 6 showsthat these proteins have substantial similarity to vicilin seed-strorageproteins from cocoa and cotton. Stars show positions of conservedidentities and dots show positions of conserved similarities.Examination of the protein sequences revealed that the exact MiAMP2csequence is found within the translated protein sequence of clone 3 atamino acid positions 118 to 164 (see FIG. 6); clones 1 and 2 containedsequences differing from MiAMP2c by 2 residues and 3 residues,respectively, out of 47 amino acids total in the MiAMP2c sequence.

[0124] The translation products of the full-length clones (i.e., clones1 and 2) consist of a short signal peptide from residues 1 to 28, ahydrophilic region from residues 29 to ˜246, and then two segmentsstretching from residues ˜246 to 666 with a stretch of acidic residuesseparating them at positions 542-546.

[0125] Significantly, the hydrophilic region containing the sequence forMiAMP2c, also contains 3 additional segments which are very similar toMiAMP2 (termed MiAMP2a, b and d). These 4 segments (found betweenresidues 28 and ˜246) are separated by stretches in which approximatelyfour out of five residues are acidic (usually glutamic acid). Theseacidic stretches occur at positions 64-68, 111-115, 171-174, and 241-246and appear to delineate processing sites for cleavage of the 666-residuepreproprotein into smaller functional fragments (acidic stretchesdelineating cleavage sites are shown as bold characters in FIG. 6). Allfour MiAMP2-like segments of the protein contain 2 doublets of cysteineresidues separated by 10-12 residues to give the following patternC-X-X-X-C-(10-12X)-C-X-X-X-C where X is any amino acid, and C iscysteine. All four segments are expected to form helix-turn-helix motifsas decribed in Example 8 above. It is clear that the cysteines in theselocations will form disulfide bridges that stabilize the structure ofthe proteins by holding the two helical portions together.

[0126] The predicted helix-turn-helix motifs can be further stabilizedin several ways. The first method of stabilization is exemplified insegments 1 and 3 (i.e., residues 29-63 and 118-170, respectively, of the666-residue Macadamia vicilin-like protein). These segments is the arestabilized by a hydrophobic ring-stacking interaction between twoaromatic residues (one on each α-helical segment); this is normallyaccomplished with tyrosine residues but phenylalanine is also used. Aswith the cysteine residues, the location of these aromatic residues inthe predicted α-helical segments is critical if they are to offerstabilization to the helix-turn-helix structure. In segments 1 and 3,the aromatic residues are 2 and 3 residues removed from the cysteinedoublets as shown here: Z-X-X-C-X-X-X-C-(10-12X)-C-X-X-X-C-X-X-X-Z whereC is cysteine and Z is usually tyrosine but can be substituted withphenylalanine as is done in segment 1.

[0127] The second way to stabilize the helix-turn-helix fragment is byusing an added disulfide bridge as seen in fragment 2 (residues 71-110).This is accomplished by placing additional cysteine residues 2 and 3residues removed from the cysteine doublets as shown here:nX-C-X-X-C-X-X-X-C-(10-12X)-C-X-X-X-C-X-X-X-C-nX. This is the onlyreport that the inventors know of where a helix-turn-helix domain in anantimicrobial protein is stabilized by three disulfide bridges. Whilesegment 4 (residues 175-241) does not contain the extra disulfide bridgeor the hydrophobic ring-stacking stabilization, it is probablystabilized by means of weaker ionic and or hydrogen bondinginteractions.

EXAMPLE 10 Vectors for Liquid Culture Expression of MiAMP2 andHomologues

[0128] PCR primers flanking the nucleotide region coding for MiAMP2cwere engineered to contain restriction sites for Nde I and Bam HI(corresponding to the 5′ and 3′ ends of the coding region, respectively;Primer JPM31 sequence: 5′ A CAC CAT ATG CGA CAA CGT GAT CC 3′; PrimerJPM32 sequence: 3′ C GTT GTT TTC TCT ATT CCT AGG GTT G 5′, SEQ ID NOS:14 and 15). These primers were then used to amplify the coding region ofMiAMP2c DNA. The PCR product from this amplification was then digestedwith Nde I and Bam HI and ligated into a pET17b vector (Novagen/Studier,F. W. et al. [1986] J Mol. Biol. 189:113) with the coding regionin-frame to produce the vector pET 17-MiAMP2c.

[0129] A similar approach to the one above was used to construct vectorscarrying the coding sequences of MiAMP2c homologues (i.e. MiAMP2a, b,and d as well as Tc AMP1, and TcAMP2). To construct the expressionvectors for fragments a, b and d in MiAMP2 clone 1, specific PCR primersincorporating the Nde I and Bam HI sites were designed to amplify thefragments of interest. The products were then digested with theappropriate restriction enzymes and ligated into the Nde I/Bam HI sitesof a pET16b vector [Novagen] containing a His tag and a Factor Xacleavage site (amino acid sequence MGHHH HHHHH HHSSG HIEGR HM, SEQ IDNO: 16). The protein products expressed from the pET16b vector is afusion to the antimicrobial protein. The coding sequences forMiAMP2-like subunits from cocoa (FIG. 4, TcAMP1 and TcAMP2) wereobtained from the published DNA sequence of the cocoa vicilin gene(Spencer, M. E. and Hodge R. [1992] Planta 186:567-576). Two MiAMP2-likefragments within the cocoa vicilin gene were located at the 5′ end(corresponding to the residues shown in FIG. 4), and two sets ofcomplimentary oligonucleotides corresponding to the desired codingsequences were designed. The complimentary oligonucleotides (90 to ˜100bases) corresponding to each cocoa subunit contained a 20 bp overlap andalso contained the Nde I and Bam HI restriction endonuclease cut sites.For TcAMP, the following nucleotides were synthesised: TcAMP1 forwardoligo 5′ GGGAATTCCA TATGTATGAG CGTGATCCTC GACAGCAATA CGAGCAATGCCAGAGGCGAT GCGAGTCGGA AGCGACTGAA GAAAGGGAGC 3′; TcAMP1 reverse oligo5′ GAAGCGACTG AAGAAAGGGA GCAAGAGCAG TGTGAACAAC GCTGTGAAAG GGAGTACAAGGAGCAGCAGA GACAGCAATA GGGATCCACA C 3′. For TcAMP2, the followingoligonucleotides were used: TcAMP2 forward oligo 5′ GGGAATTCCATATGCTTCAA AGGCAATACC AGCAATGTCA AGGGCGTTGT CAAGAGCAAC AACAGGGGCAGAGAGAGCAG CAGCAGTGCC AGAGAAAATG C 3′; TcAMP2 reverse oligo5′ GTGTGGATCC CTAGCTCCTA TTTTTTTTGT GATTATGGTA ATTCTCGTGC TCGCCTCTCTCTTGTTCCTT ATATTGCTCC CAGCATTTTC TCTGGCACTG CT 3′.

[0130] The oligonucleotide sets were added to individual PCRamplification reactions in order make individual PCR fragmentscontaining the desired coding region. Since initial PCR amplificationsgave fuzzy bands, reamplification of the original products was carriedout using new 20mer primers (complimentary to the 5′ends of the forwardand reverse oligonucleotides shown above) designed to amplify the entirecoding region of the cocoa subunits. Once amplified, the PCR productswere restriction digested with the appropriate enzymes and ligated intothe vector pET16b as above. This procedure was carried out for bothcocoa fragments with similarities to MiAMP2c (shown in FIG. 4).

EXAMPLE 11 Expression in E. coli and Purification of MiAMP2c andHomologues

[0131] Starter cultures (50 ml) of E. coli strain BL21 (Grodberg, J.[1988] J. Bacteriol. 170:1245) transformed with the appropriate pETconstruct (Example 10) were added to 500 ml of NZCYM media (CurrentProtocols in Molecular Biology, supra) and cultured to an opticaldensity of 0.6 (600 nm) and induced with the addition of 0.4 or 1.0 mMIPTG depending on whether pET17b (containing a T7 promoter) or pET16b(containing a His tag fusion and a T7 promoter/lac operator) vector wasbeing used. After cells were induced, cultures were allowed to grow for4 hours before harvesting. Aliquots of the growing cultures were removedat timed intervals and protein extracts run on an SDS-PAGE gel to followthe expression levels of MiAMP2 and homologues in the cultures.Fragments being expressed with a Histidine tag (i.e., in the pET16bvector), were harvested by centrifuging induced cell cultures at 5000gfor 10 minutes. Cell pellets were resuspended and broken by stirring forone hour in 6 M Guanidine-HCl, buffered with 100 mM sodium phosphate and10 mM Tris at pH 8.0. Broken cell suspensions were centrifuged at10,000g for 20-30 minutes to settle the cellular debris. Supernatantswere removed to fresh tubes and 500 mg of Ni-NTA fast flow resin(Qiagen) was added to each supternatant. After gentle mixing at 4° C.for 30-60 minutes, the suspension was loaded into a small column, rinsedtwo times with 8 M Urea (pH 8.0 and then pH 6.3) and subsequently, theprotein was eluted using 8 M Urea pH 4.5. Protein fractions thusobtained were substantially pure but were further purified using an9.3×250 mm C2/C18 reverse phase column (Pharmacia) and 75 minutegradient from 5% to 50% acetonitrile (0.1% TFA) flowing at 3 ml/min(data not shown).

[0132] All of the MiAMP2c homologues (except MiAMP2c which was expressedin pET17b) were expressed in the pET16b vector containing the Histidinetag. While induction of the MiAMP2c culture proceded as above, the restof the purification was somewhat different. In this case,MiAMP2c-expressing cells were harvested by centrifugation but were thenresuspended in phosphate buffer (100 mM, pH 7.0 containing 10 mM EDTAand 1 mM PMSF) and broken open using a French press instrument. Cellulardebris containing MiAMP2c inclusion bodies was solubilized using a 6 MGuanidine-HCl, 10 mM MES pH 6.0 buffer. Soluble material was thenrecovered after centrifugation to remove insoluble debris remaining fromthe solubilization step. Guanidine-HCl soluble material was thendialyzed against 10 mM MES pH 6.0 containing PMSF (1 mM) and EDTA (10mM). Cation-exchange fractionation was carried out as described inExample 3 except on a smaller scale after the dialysis step.Subsequently, the major eluting protein from the cation-exchange column,which was MiAMP2c, was then further purified using reverse phase HPLC asdescribed in Example 3.

[0133]FIG. 9 shows the SDS-PAGE gel analysis of the various purificationstages obtained following induction with IPTG and subsequentpurification of expressed proteins. Samples analysed during the TcAMP1purification were are as follows: lane 1, molecular weight markers; lane2, Ni-NTA non-binding fraction; lane 3, rinse of Ni-NTA resin with pH 8urea; lane 4, rinse of Ni-NTA resin with pH 6.3 urea; lane 5, elution ofTcAMP1 with pH 4.5 urea; and lane 6, second elution of TcAMP1 with pH4.5 urea. TcAMP2 was purified in a similar manner and was also subjectedto reverse-phase HPLC to further purify the fraction eluting from theNi-NTA resin. FIG. 10 shows the reverse phase purification of cocoasubunit number 2 (TcAMP2).

[0134] SDS-PAGE gel analysis of the MiAMP2a, b, and d fragmentpurification is shown in the second panel of FIG. 9. Lane contents areas follows: lane 1, molecular weight markers; lane 2, MiAMP2apre-induced cellular extractp; lane 3, MiAMP2a IPTG induced cellularextract; lane 4, MiAMP2a Ni-NTA non-binding fraction; lane 5, MiAMP2aelution from Ni-NTA; lane 6, MiAMP2b pre-induced cellular extract; lane7, MiAMP2b IPTG induced cellular extract; lane 8, MiAMP2b Ni-NTAnon-binding fraction; lane 9, MiAMP2b elution from Ni-NTA; lane 10,MiAMP2d pre-induced cellular extract; lane 11, MiAMP2d IPTG inducedcellular extract; lane 12, MiAMP2d Ni-NTA non-binding fraction; and lane13, MiAMP2d elution from Ni-NTA.

[0135] Using the vectors described in Example 10, MiAMP2c, and 5homologues (i.e., MiAMP2a, MiAMP2b, MiAMP2d, TcAMP1 and TcAMP2) were allexpressed, purified and tested for antimicrobial activity. The approachtaken above can be applied to all of the antimicrobial fragmentsdescribed in FIG. 4. Purified fragments can then be tested for specificinhibition agains microbial pathogens of interest.

EXAMPLE 12 Detection of MiAMP2 Homologues in other Species UsingAntibodies Raised to MiAMP2c

[0136] Rabbits were immunised intramuscularly according to standardprotocols with MiAMP2 conjugated to diphtheria toxoid suspended inFruends incomplete adjuvent. Serum was harvested from the animals atregular intervals after giving the animal added doses of MiAMP2 adjuventto boost the immune response. Approximately 100 ml of serum werecollected and used for screening of crude extracts obtained from severalplant seeds. One hundred gram quantities of seeds were ground andextracted to obtain a crude extract as in Example 1. Aliquots of proteinwere separated on SDS-PAGE gels and the gels were then blotted ontonitrocellulose membrane for subsequent detection of antibody reactingproteins. The membranes were incubated with MiAMP2c rabbit primaryantibodies, washed and then incubated with alkalinephosphatase-conjugated goat anti-rabbit IgG for colorimetric detectionof antigenic bands using the chemical 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium substrate system (Schleicher andSchuell). FIG. 11 shows that various other species containimmunologically-related proteins of similar size to MiAMP2c. Lanes 1-15contain the extracts from the following species: 1) Stenocarpussinuatus, 2) Stenocarpus sinuatus ({fraction (1/10)} loading), 3) Restiotremulus, 4) Mesomalaena tetragona, 5) Nitraria billardieri, 6)Petrophile canescens, 7) Synaphae acutiloba, 8) Dryandra formosa, 9)Lambertia inermis, 10) Stirlingia latifolia, 11) Xylomelumangustifolium, 12) Conospermum bracteosum, 13) Conospermumtriplinernium, 14) Molecular weight marker, 15) Macacamia integrifoliapure MiAMP2c. Lanes 1-13 contain a variety of species, some of whichshow the presence of antigenically related proteins of a similar size toMiAMP2c. Other bands exhibiting higher molecular weights probablyrepresent the larger precursor seed storage proteins from which theantimicrobial proteins are derived. Antigenically-related proteins canbe seen in lanes 1, 2, 4, 6, 7, 8, 9, and 11-13.

[0137] Bioassays were also performed using crude extracts from variousProteaceae species. Specifically, extracts from Banksia robur, Banksiacanei, Hakea gibbosa, Stenocarpus sinuatus, and Stirlingia latifoliahave all been shown to exhibit antimicrobial activity. This activity mayderive from MiAMP2 homologues since these species are related toMacadamia.

EXAMPLE 13 Purification of MiAMP2c Homologues in Another Species UsingAntibodies Raised to MiAMP2c

[0138] Based on the detection of immunologically related proteins inother species of the family Proteaceae and the presence of antimicrobialactivity in crude extracts, Stenocarpus sinuatis was chosen for a largescale fractionation experiment in an attempt to isolate MiAMP2chomologues. Five kg of S. sinuatus seed was frozen in liquid nitrogenand ground in a food processor (Big Oscaar Sunbeam). The ground seed wasimmediately placed into 12 L of 50 mM H₂SO₄ extraction buffer andextracted at 4° C. for 1 hour with stirring. The slurry was thencentrifuged for 20 min at 10,000 g to remove particulate matter. Thesupernatant was then adjusted to pH 9 using a 50 mM ammonia solution.PMSF and EDTA were added to final concentrations of 1 and 10 mMrespectively.

[0139] The crude protein extract was applied to an anion exchange column(Amberlite IRA-938, Rohm and Haas) (3 cm×90 cm) equilibrated with 50 mMNH₄Ac pH 9.0 at a flow rate of 40 ml/min. The unbound protein comprisingthe basic protein fraction was collected and used in the subsequentpurification steps.

[0140] The basic protein fraction was adjusted to pH 5.5 with aceticacid and then applied at 10 ml/minute over 12 h to a SP-Sepharose FastFlow (Pharmacia) Column (5 cm×60 cm) pre-equilibrated with 25 mMammonium acetate. The column was then washed for 3.5 h with 25 mMAcetate pH 5.5. Elution of bound proteins was achieved by applying alinear gradient of NH₄Ac from 25 mM to 2.0 M (pH 5.5) at 10 ml/min over10 h. Absorbance of the eluate was observed at 280 nm and 100 mlfractions collected (see FIG. 12).

[0141] Cation-exchange fractions that cross-reacted with the antiserum(fractions 14-28, FIG. 12) were then further purified by reverse phasechromatography. Cross-reacting fractions were loaded onto a 7 μm Cl 8reverse phase column (Brownlee) equilibrated with 90% H₂O, 10%acetonitrile and 0.1% Trifluoroacetic acid (TFA)(=100%A). Bound proteinswere eluted with a linear gradient from 100%A to 100%B (5% H₂O, 95%acetonitrile, 0.08% TFA). The absorbance of the eluted proteins wasmonitored at 214 nm and 280 nm. The eluted proteins were dried undervacuum and resuspended in water three times to remove traces of TFA fromthe samples. Reverse phase protein elution fractions 20 to 61 wereanalysed by pooling 2 adjacent fractions and performing a western blotanalysis (see FIG. 13). Fractions 22-41 gave a weak positive reactionand fractions 42-57 gave a strong positive reaction to the anti-MiAMP2cantiserum. Fractions that showed antifungal activity against S.sclerotiorum at 50 μg/ml and 10 μg/ml are indicated by arrows on thechromatogram.

[0142] Using the approach above, several active fractions (termed SsAMP1and SsAMP2) were obtained which were assessed for their antifungalactivity against Sclerotinia sclerotiorum, Alternaria brassicola,Leptosphaeria maculans, Verticilium dahliae and Fusarium oxysporum.Bioassays were carried out as described in Example 2 and results shownin Example 15. Another fragment which reacted with MiAMP2 antiserum waspurified and sequenced (SsAMP3) but insufficient protein was availablefor characterisation of antimicrobial activity. Partial sequencesobtained from these proteins are shown in FIG. 4 (SEQ ID NOS: 26, 27 and28). Full sequencing of the peptides or cloning of cDNAs encoding theseed storage proteins from this species will reveal the extent ofhomology between these peptides and MiAMP2-series homologues.

EXAMPLE 14 Synthesis of Small Fragments of MiAMP2c

[0143] In an effort to determine if the full MiAMP2c molecule wasabsolutely necessary for the protein to exhibit antimicrobial activity,two separate peptides were chemically synthesized by Auspep Pty. Ltd.(Australia). For each peptide, the cysteine residues were changed toalanine residues so that disulfide bonds were no longer capable of beingformed between two separate protein chains. Tyrosine residues were alsochanged to alanine since it was expected that tyrosine also participatedin the helix-turn-helix stabilization and this would not be needed inthe synthetic peptides lacking one of the helices. Alanine is alsofavorable to the formation of alpha-helices so it should not interferewith the native helical structure to a large degree. Peptide one iscomprised of 22 amino acids from 118 to 139 in the amino acid sequenceof clone 3 (sequence: RQRDP QQQAE QAQKR AQRRE TE, SEQUENCE ID NO: 9).Peptide 2 is 25 amino acids in length and runs from 140 to 164 in clone3 (sequence: PRHMQ IAQQR AERRA EKEKR KQQKR, SEQ ID NO: 10). Peptides 1and 2 are labeled MiAMP2c pep1 and MiAMP2c pep2 respectively. Thesepeptides were resuspended in Milli-Q water and bioassayed against anumber of fungi. As seen in Table 2, peptide 2 has inhibitory activityagainst a variety of fungi whereas peptide 1 exhibited little or noactivity. Mixtures of peptide 1 and peptide 2 exhibit similar levels ofactivity as seen with peptide 2 alone indicating that only peptide 2 isexhibiting activity. The fact that peptide 2 exhibits antimicrobialactivity in the absence of the helix-turn-helix structure exhibited byMiAMP2c reveals that the helix-turn-helix structure is not absolutelynecessary for the peptides to retain activity. Nevertheless, peptide 2did not exhibit the same degree of activity on a molar basis as MiAMP2c(whole fragment) indicating that the helix-turn-helix structure isimportant for maximal expression of antimicrobial activity by thefragments involved. It is also expected that the helix-turn-helixstructure will confer greater stability to the MiAMP2 homologues, thusrendering these proteins less susceptible to proteolytic cleavage andother forms of degradation. Greater stability would lead to maintainingantimicrobial activity over a longer period of time.

EXAMPLE 15 Antifungal Activity of MiAMP2c Homologues and Fragment(s)

[0144] MiAMP2c and each of the various MiAMP2 homologues were testedagainst a variety of fungi as concentrations ranging from 2 to 50 μg/ml.Table 1 shows the IC₅₀ value of pure MiAMP2c against various fungi andbacteria. In the table, the “>50” indicates that 50% inhibition of thefungus was not achieved at 50 μg/ml which was the highest concentrationtested. The abbreviation “ND” indicates that the test was not performedor that results could not be interpreted. The antimicrobial activity ofMiAMP2c was also tested in the presence bf 1 mM Ca²⁺ in the test mediumand the IC₅₀ values for these tests are given in the right-hand column.As can be seen in the table, the inhibitory activity of MiAMP2c isgreatly reduced (although not eliminated) in the presence of Ca²⁺. TABLE1 Concentrations of MiAMP2c at which 50% inhibition of growth wasobserved Organism IC₅₀ (μg/ml) IC₅₀ + Ca²⁺ (μg/ml) Alternaria helianthi 5-10 ND Candida albicans >50 >50 Ceratocystis paradoxa 20-50 >50Cercospora nicotianae  5-10     5-10  Clavibacter michiganensis 50 >50Chalara elegans 2-5    10-20 Fusarium oxysporum 10    20-50 Sclerotiniasclerotiorum 20-50 >50 Phytophthora cryptogea  5-10    10-25Phytophthora parasitica nicotiana 10-20 >50 Verticillium dahliae 5-10 >50 Ralstonia solanacearum >50 >50 Pseudomonas syringaetabaci >50 >50 Saccharomyces cerevisiae 20-50 >50 Escherichia coli >50>50

[0145] Table 2 shows the the antimicrobial activity of varioushomologues and fragments of MiAMP2c. In the table, the followingabbreviations are used: Ab, Alternaria brassicola; Cp: Ceratocystisparadoxa; Foc: Fusarium oxysporum; Lm: Leptosphaeria maculans; Ss:Sclerotinia sclerotiorum; Vd: Verticillium dahlias. The “>50” indicatesthat concentrations higher than 50 μg/ml were not tested so that an IC₅₀value could not be established. A blank space indicates that the testwas not performed or that results could not be interpreted.

[0146] The TcAMP1 and 2 used for the results presented in Table 2 werederived from cocoa vicilin (Examples 10 and 11). SsAMP1 and 2 showreactivity with MiAMP2c antibodies and also exhibit antimicrobialactivity as seen in the table below. The versions of MiAMP2a, b and d aswell as TcAMP1 and TcAMP2 tested in the bioassays all contain a His tagfusion resulting from expression in the vector pET16b. MiAMP2c pep1 and2 are the N and C terminal regions, respectively, of MiAMP2cantimicrobial peptide as specified in Example 14 above. Theconcentration value listed for ‘MiAMP2c pep1+2’ is the concentration ofeach individual peptide in the mixture. It should be remembered thatMiAMP2c pep1 and pep2 are both about ½ the size of MiAMP2c; comparisonsof the activity of these peptides with the MiAMP2c protein should,therefore, be made on a molar basis rather than on a strict μg/mlconcentration basis. Peptides were only tested in media A which did notcontain added Ca²⁺. TABLE 2 IC₅₀ values (μg/ml) of MiAMP2 relatedproteins against various fungi Peptide Fungus used in bioassy tested AbCp Foc Lm Ss Vd MiAMP2a  5-10 2.5-5  5-10 MiAMP2b 2.5 2.5  5-10 MiAMP2c20-50 10 20-50  5-10 MiAMP2d 5 2.5  5-10 MiAMP2c 100 >50 pep1 MiAMP2c10-20   10-20 50 10-20 pep2 MiAMP1c 10-25 50 pep1 + 2 TcAMP1 10  5-10  2-5 10  5-20 TcAMP2  5-10  5-10   2-5 5  5-20 SsAMP1 20-50   20-5020-50 10-20 SsAMP2 20-50 >50 >50 >50 >50

[0147] It is worthy of note that while the TcAMP1 and 2 sequences arereadily available in the public data bases, no antimicrobial activityhad ever been assigned to them. These sequences were derived from muchlarger proteins involved in seed storage functions. The inventors havethus described a completely new activity for a small portion of theoverall cocoa vicilin molecules. The activity of cotton fragments 1, 2,and 3 has been exemplified by other authors (Chung, R. P. T. et al.[1997] Plant Science 127:1-16).

EXAMPLE 16 Construction of the Plant Transfomation Vector PCV91-MiAMP2c

[0148] The expression vector pPCV91-MiAMP2c (FIG. 14) contains the fullcoding region of the MiAMP2c (Example 7) DNA flanked at it 5′ end by thestrong constitutive promoter of 35S RNA from the cauliflower mosaicvirus (pCaMV35S) (Odel et al., [1985] Nature 313: 810-812) with aquadruple-repeat enhancer element (e-35S) to allow for hightranscriptional activity (Kay et al. [1987] Science 236:1299-1302). Thecoding region of MiAMP2c DNA is flanked at its 3′ end by thepolyadenylation sequence of 35S RNA of the cauliflower mosaic virus(pA35S). The plasmid backbone of this vector is the plasmid pPCV91(Walden, R. et al. [1990] Methods Mol. Cell. Biol. 1:175-194). Theplasmid also contains other elements useful for plant transformationsuch as an ampicillin resistance gene (bla) and a hygromycin resistancegene (hph) driven by the nos promoter (pnos). These and other featuresallow for selection in various cloning and transformation procedures.The plasmid pPCV91-MiAMP2c was constructed as follows: A cloned fragmentencoding MiAMP2c (Example 7) was digested using restriction enzymes torelease the MiAMP2c gene fragment containing a synthetic leadersequence. The binary vector pPCV91 was digested with the restrictionenzyme Bam HI. Both the MiAMP2c DNA fragment containing and the binaryvector were ligated using T4 DNA ligase to produce pPCV9 1-MiAMP2cbinary vector for plant transformation (FIG. 12).

[0149] Using this approach, other homologues of MiAMP2c can be expressedin plants. Not only can individual homologues be expressed, but they maybe expressed in combination with other proteins as fusion proteins or asportions of larger precursor proteins. For example, it is possible toexpress the N-terminal region of MiAMP2 clone 1 (amino acids 1 to ≈246)which contains a signal peptide and the hydrophilic region containingfour antimicrobial segments. Transgenic plants can then be assessed toexamine whether the individual fragments are being processed into theexpected fragments by the processing machinery already present in theplant cells. It is also possible to express the entire MiAMP2 clone 1(amino acids 1 to 666) and to examine the processing of the entireprotein when expressed in transgenic plants. Homologous regions fromother sequences can also be used in multiple combinations with, forexample, ten (10) or more MiAMP2-like fragments expressed as one largefusion protein with acidic cleavage sites located as proper locationsbetween each of the fragments. As well as linking MiAMP2 fragmentstogether, it would also be possible to link MiAMP2 fragments to otheruseful proteins for expression in plants.

EXAMPLE 17 Transgenic Plants Expressing MiAMP2c (or Related Fragments)

[0150] The disarmed Agrobacterium tumefaciens strain GV3101 (pMP90RK)(Koncz, Cs.[1986] Mol. Gen. Genet. 204:383-396) was transformed with thevector pPCV91-MiAMP2c (Example 16) using the method of Walkerpeach etal. (Plant Mol. Biol. Manual B1:1-19 [1994]) adapted from Van Haute etal (EMBO J. 2:411-417 1983]).

[0151] Tobacco transformation was carried out using leaf discs ofNicotiana tabacum based on the method of Horsch et al. (Science227:1229-1231 [1985]) and co-culturing strains containingpPCV91-MiAMP2c. After co-cultivation of Agrobacterium and tobacco leafdisks, transgenic plants (transformed with pPCV91-MiAMP2c) wereregenerated on media containing 50 μg/ml hygromycin and 500 μg/mlCefotaxime. These transgenic plants were analysed for expression of thenewly-introduced genes using standard western blotting techniques (FIG.15). FIG. 15 shows a western blot of extracts from trangenic tobaccocarrying the construct for MiAMP2c from example 16. Lane 1 contains pureMiAMP2c as a standard, lanes 2 and 3 contain extracts from transgenicplants canying the pPCV9 1-MiAMP2c construct. As can be see in thefigure, faint bands are present at approximately the correct molecularweight, indicating that the transgenic plants appear to be expressingthe MiAMP2c protein. Plants capable of constitutive expression of theintroduced genes may be selected and self-pollinated to give seed. Flseedlings of the transgenic plants may be further analysed.

EXAMPLE 18 MiAMP2c Homologues

[0152] Every homologue of MiAMP2c that has been tested has exhibitedsome antimicrobial activity. This evidence indicates that otherhomologues will also exhibit antimicrobial activity. These homologuesinclude fragments from 1) peanut (Burks, A. W. et al. [1995] J. Clin.Invest. 96 (4), 1715-1721), 2) maize (Belanger, F. C. and Kriz, A.L.[1991] Genetics 129 (3), 863-872), 3) barley (Heck, G. R. et al.[1993] Mol. Gen. Genet. 239 (1-2), 209-218), and 4) soybean (Sebastiani,F. L. et al. [1990] Plant Mol. Biol. 15(1), 197-201). (see SEQ ID NOS:21,22,24, and 25). Other sequences derived from seed storage proteins ofthe 7S class are also expected to yield homologues of MiAMP2 proteins.

1 40 1 666 PRT Macadamia integrifolia 1 Met Ala Ile Asn Thr Ser Asn LeuCys Ser Leu Leu Phe Leu Leu Ser 1 5 10 15 Leu Phe Leu Leu Ser Thr ThrVal Ser Leu Ala Glu Ser Glu Phe Asp 20 25 30 Arg Gln Glu Tyr Glu Glu CysLys Arg Gln Cys Met Gln Leu Glu Thr 35 40 45 Ser Gly Gln Met Arg Arg CysVal Ser Gln Cys Asp Lys Arg Phe Glu 50 55 60 Glu Asp Ile Asp Trp Ser LysTyr Asp Asn Gln Glu Asp Pro Gln Thr 65 70 75 80 Glu Cys Gln Gln Cys GlnArg Arg Cys Arg Gln Gln Glu Ser Gly Pro 85 90 95 Arg Gln Gln Gln Tyr CysGln Arg Arg Cys Lys Glu Ile Cys Glu Glu 100 105 110 Glu Glu Glu Tyr AsnArg Gln Arg Asp Pro Gln Gln Gln Tyr Glu Gln 115 120 125 Cys Gln Lys HisCys Gln Arg Arg Glu Thr Glu Pro Arg His Met Gln 130 135 140 Thr Cys GlnGln Arg Cys Glu Arg Arg Tyr Glu Lys Glu Lys Arg Lys 145 150 155 160 GlnGln Lys Arg Tyr Glu Glu Gln Gln Arg Glu Asp Glu Glu Lys Tyr 165 170 175Glu Glu Arg Met Lys Glu Glu Asp Asn Lys Arg Asp Pro Gln Gln Arg 180 185190 Glu Tyr Glu Asp Cys Arg Arg Arg Cys Glu Gln Gln Glu Pro Arg Gln 195200 205 Gln His Gln Cys Gln Leu Arg Cys Arg Glu Gln Gln Arg Gln His Gly210 215 220 Arg Gly Gly Asp Met Met Asn Pro Gln Arg Gly Gly Ser Gly ArgTyr 225 230 235 240 Glu Glu Gly Glu Glu Glu Gln Ser Asp Asn Pro Tyr TyrPhe Asp Glu 245 250 255 Arg Ser Leu Ser Thr Arg Phe Arg Thr Glu Glu GlyHis Ile Ser Val 260 265 270 Leu Glu Asn Phe Tyr Gly Arg Ser Lys Leu LeuArg Ala Leu Lys Asn 275 280 285 Tyr Arg Leu Val Leu Leu Glu Ala Asn ProAsn Ala Phe Val Leu Pro 290 295 300 Thr His Leu Asp Ala Asp Ala Ile LeuLeu Val Ile Gly Gly Arg Gly 305 310 315 320 Ala Leu Lys Met Ile His HisAsp Asn Arg Glu Ser Tyr Asn Leu Glu 325 330 335 Cys Gly Asp Val Ile ArgIle Pro Ala Gly Thr Thr Phe Tyr Leu Ile 340 345 350 Asn Arg Asp Asn AsnGlu Arg Leu His Ile Ala Lys Phe Leu Gln Thr 355 360 365 Ile Ser Thr ProGly Gln Tyr Lys Glu Phe Phe Pro Ala Gly Gly Gln 370 375 380 Asn Pro GluPro Tyr Leu Ser Thr Phe Ser Lys Glu Ile Leu Glu Ala 385 390 395 400 AlaLeu Asn Thr Gln Thr Glu Lys Leu Arg Gly Val Phe Gly Gln Gln 405 410 415Arg Glu Gly Val Ile Ile Arg Ala Ser Gln Glu Gln Ile Arg Glu Leu 420 425430 Thr Arg Asp Asp Ser Glu Ser Arg His Trp His Ile Arg Arg Gly Gly 435440 445 Glu Ser Ser Arg Gly Pro Tyr Asn Leu Phe Asn Lys Arg Pro Leu Tyr450 455 460 Ser Asn Lys Tyr Gly Gln Ala Tyr Glu Val Lys Pro Glu Asp TyrArg 465 470 475 480 Gln Leu Gln Asp Met Asp Leu Ser Val Phe Ile Ala AsnVal Thr Gln 485 490 495 Gly Ser Met Met Gly Pro Phe Phe Asn Thr Arg SerThr Lys Val Val 500 505 510 Val Val Ala Ser Gly Glu Ala Asp Val Glu MetAla Cys Pro His Leu 515 520 525 Ser Gly Arg His Gly Gly Arg Gly Gly GlyLys Arg His Glu Glu Glu 530 535 540 Glu Asp Val His Tyr Glu Gln Val ArgAla Arg Leu Ser Lys Arg Glu 545 550 555 560 Ala Ile Val Val Leu Ala GlyHis Pro Val Val Phe Val Ser Ser Gly 565 570 575 Asn Glu Asn Leu Leu LeuPhe Ala Phe Gly Ile Asn Ala Gln Asn Asn 580 585 590 His Glu Asn Phe LeuAla Gly Arg Glu Arg Asn Val Leu Gln Gln Ile 595 600 605 Glu Pro Gln AlaMet Glu Leu Ala Phe Ala Ala Pro Arg Lys Glu Val 610 615 620 Glu Glu SerPhe Asn Ser Gln Asp Gln Ser Ile Phe Phe Pro Gly Pro 625 630 635 640 ArgGln His Gln Gln Gln Ser Pro Arg Ser Thr Lys Gln Gln Gln Pro 645 650 655Leu Val Ser Ile Leu Asp Phe Val Gly Phe 660 665 2 2171 DNA Macadamiaintegrifolia sig_peptide (1)...(85) mat_peptide (86)...(1999) 2atggcgatca atacatcaaa tttatgttct cttctctttc tcctttcact cttccttctg 60tctacgacag tgtctcttgc tgaaagtgaa tttgacaggc aggaatatga ggagtgcaaa 120cggcaatgca tgcagttgga gacatcaggc cagatgcgtc ggtgtgtgag tcagtgcgat 180aagagatttg aagaggatat agattggtct aagtatgata accaagagga tcctcagacg 240gaatgccaac aatgccagag gcgatgcagg cagcaggaga gtggcccacg tcagcaacaa 300tactgccaac gacgctgcaa ggaaatatgt gaagaagaag aagaatataa ccgacaacgt 360gatccacagc agcaatacga gcaatgtcag aagcactgcc aacggcgcga gacagagcca 420cgtcacatgc aaacatgtca acaacgctgc gagaggagat atgaaaagga gaaacgtaag 480caacaaaaga gatatgaaga gcaacaacgt gaagacgaag agaaatatga agagcgaatg 540aaggaagaag ataacaaacg cgatccacaa caaagagagt acgaagactg ccggaggcgc 600tgcgaacaac aggagccacg tcagcagcac cagtgccagc taagatgccg agagcagcag 660aggcaacacg gccgaggtgg cgatatgatg aaccctcaga ggggaggcag cggcagatac 720gaggagggag aagaggagca aagcgacaac ccctactact tcgacgaacg aagcttaagt 780acaaggttca ggaccgagga aggccacatc tcagttctgg agaacttcta tggtagatcc 840aagcttctac gcgcactaaa aaactatcgc ttggtgctcc tcgaggctaa ccccaacgcc 900ttcgtgctcc ctacccactt ggatgcagat gccattctct tggtcatagg agggagagga 960gccctcaaaa tgatccacca cgacaacaga gaatcctaca acctcgagtg tggagacgta 1020atcagaatcc cagctggaac cacattctac ttaatcaacc gagacaacaa cgagaggctc 1080cacatagcca agttcttaca gaccatatcc actcctggcc aatacaagga attcttccca 1140gctggaggcc aaaacccaga gccgtacctc agtaccttca gcaaagagat tctcgaggct 1200gcgctcaaca cacaaacaga gaagctgcgt ggggtgtttg gacagcaaag ggagggagtg 1260ataattaggg cgtcacagga gcagatcagg gagttgactc gagatgactc agagtcacga 1320cactggcata taaggagagg tggtgaatca agcaggggac cttacaatct gttcaacaaa 1380aggccactgt actccaacaa atacggtcaa gcctacgaag tcaaacctga ggactacagg 1440caactccaag acatggactt atcggttttc atagccaacg tcacccaggg atccatgatg 1500ggtcccttct tcaacactag gtctacaaag gtggtagtgg tggctagtgg agaggcagat 1560gtggaaatgg catgccctca cttgtcggga agacacggcg gccgcggtgg aggaaaaagg 1620catgaggagg aagaggatgt gcactatgag caggttagag cacgtttgtc gaagagagag 1680gccattgttg ttctggcagg tcatcccgtc gtcttcgttt catccggaaa cgagaacctg 1740ctgctttttg catttggaat caatgcccaa aacaaccacg agaacttcct cgcggggaga 1800gagaggaacg tgctgcagca gatagagcca caggcaatgg agctagcgtt tgccgctcca 1860aggaaagagg tagaagagtc atttaacagc caggaccagt ctatcttctt tcctgggccc 1920aggcagcacc agcaacagtc gccccgctcc accaagcaac aacagcctct cgtctccatt 1980ctggacttcg ttggcttcta aagttccaca aaaaagagtg tgttatgtag tataggttag 2040tagctcctag ctcggtgtat gagagtggta agagactaag acgctaaatc cctaagtaac 2100taacctggcg agcttgcgtg tatgcaaata aagaggaaca gctttccaac tttaaaaaaa 2160aaaaaaaaaa a 2171 3 666 PRT Macadamia integrifolia SIGNAL (1)...(28)PEPTIDE (29)...(666) 3 Met Ala Ile Asn Thr Ser Asn Leu Cys Ser Leu LeuPhe Leu Leu Ser 1 5 10 15 Leu Phe Leu Leu Ser Thr Thr Val Ser Leu AlaGlu Ser Glu Phe Asp 20 25 30 Arg Gln Glu Tyr Glu Glu Cys Lys Arg Gln CysMet Gln Leu Glu Thr 35 40 45 Ser Gly Gln Met Arg Arg Cys Val Ser Gln CysAsp Lys Arg Phe Glu 50 55 60 Glu Asp Ile Asp Trp Ser Lys Tyr Asp Asn GlnAsp Asp Pro Gln Thr 65 70 75 80 Asp Cys Gln Gln Cys Gln Arg Arg Cys ArgGln Gln Glu Ser Gly Pro 85 90 95 Arg Gln Gln Gln Tyr Cys Gln Arg Arg CysLys Glu Ile Cys Glu Glu 100 105 110 Glu Glu Glu Tyr Asn Arg Gln Arg AspPro Gln Gln Gln Tyr Glu Gln 115 120 125 Cys Gln Glu Arg Cys Gln Arg HisGlu Thr Glu Pro Arg His Met Gln 130 135 140 Thr Cys Gln Gln Arg Cys GluArg Arg Tyr Glu Lys Glu Lys Arg Lys 145 150 155 160 Gln Gln Lys Arg TyrGlu Glu Gln Gln Arg Glu Asp Glu Glu Lys Tyr 165 170 175 Glu Glu Arg MetLys Glu Glu Asp Asn Lys Arg Asp Pro Gln Gln Arg 180 185 190 Glu Tyr GluAsp Cys Arg Arg Arg Cys Glu Gln Gln Glu Pro Arg Gln 195 200 205 Gln TyrGln Cys Gln Arg Arg Cys Arg Glu Gln Gln Arg Gln His Gly 210 215 220 ArgGly Gly Asp Leu Ile Asn Pro Gln Arg Gly Gly Ser Gly Arg Tyr 225 230 235240 Glu Glu Gly Glu Glu Lys Gln Ser Asp Asn Pro Tyr Tyr Phe Asp Glu 245250 255 Arg Ser Leu Ser Thr Arg Phe Arg Thr Glu Glu Gly His Ile Ser Val260 265 270 Leu Glu Asn Phe Tyr Gly Arg Ser Lys Leu Leu Arg Ala Leu LysAsn 275 280 285 Tyr Arg Leu Val Leu Leu Glu Ala Asn Pro Asn Ala Phe ValLeu Pro 290 295 300 Thr His Leu Asp Ala Asp Ala Ile Leu Leu Val Thr GlyGly Arg Gly 305 310 315 320 Ala Leu Lys Met Ile His Arg Asp Asn Arg GluSer Tyr Asn Leu Glu 325 330 335 Cys Gly Asp Val Ile Arg Ile Pro Ala GlyThr Thr Phe Tyr Leu Ile 340 345 350 Asn Arg Asp Asn Asn Glu Arg Leu HisIle Ala Lys Phe Leu Gln Thr 355 360 365 Ile Ser Thr Pro Gly Gln Tyr LysGlu Phe Phe Pro Ala Gly Gly Gln 370 375 380 Asn Pro Glu Pro Tyr Leu SerThr Phe Ser Lys Glu Ile Leu Glu Ala 385 390 395 400 Ala Leu Asn Thr GlnAla Glu Arg Leu Arg Gly Val Leu Gly Gln Gln 405 410 415 Arg Glu Gly ValIle Ile Ser Ala Ser Gln Glu Gln Ile Arg Glu Leu 420 425 430 Thr Arg AspAsp Ser Glu Ser Arg Arg Trp His Ile Arg Arg Gly Gly 435 440 445 Glu SerSer Arg Gly Pro Tyr Asn Leu Phe Asn Lys Arg Pro Leu Tyr 450 455 460 SerAsn Lys Tyr Gly Gln Ala Tyr Glu Val Lys Pro Glu Asp Tyr Arg 465 470 475480 Gln Leu Gln Asp Met Asp Val Ser Val Phe Ile Ala Asn Ile Thr Gln 485490 495 Gly Ser Met Met Gly Pro Phe Phe Asn Thr Arg Ser Thr Lys Val Val500 505 510 Val Val Ala Ser Gly Glu Ala Asp Val Glu Met Ala Cys Pro HisLeu 515 520 525 Ser Gly Arg His Gly Gly Arg Arg Gly Gly Lys Arg His GluGlu Glu 530 535 540 Glu Asp Val His Tyr Glu Gln Val Lys Ala Arg Leu SerLys Arg Glu 545 550 555 560 Ala Ile Val Val Pro Val Gly His Pro Val ValPhe Val Ser Ser Gly 565 570 575 Asn Glu Asn Leu Leu Leu Phe Ala Phe GlyIle Asn Ala Gln Asn Asn 580 585 590 His Glu Asn Phe Leu Ala Gly Arg GluArg Asn Val Leu Gln Gln Ile 595 600 605 Glu Pro Gln Ala Met Glu Leu AlaPhe Ala Ala Pro Arg Lys Glu Val 610 615 620 Glu Glu Leu Phe Asn Ser GlnAsp Glu Ser Ile Phe Phe Pro Gly Pro 625 630 635 640 Arg Gln His Gln GlnGln Ser Ser Arg Ser Thr Lys Gln Gln Gln Pro 645 650 655 Leu Val Ser IleLeu Asp Phe Val Gly Phe 660 665 4 2171 DNA Macadamia integrifoliasig_peptide (1)...(86) mat_peptide (87)...(1999) 4 atggcgatca atacatcaaatttatgttct cttctctttc tcctttccct cttccttctg 60 tcaacgacag tgtctcttgctgaaagtgaa tttgacaggc aggaatatga ggagtgcaaa 120 cggcaatgca tgcagttggagacatcaggc cagatgcgtc ggtgtgtgag tcagtgcgat 180 aagagatttg aagaggatatagattggtct aagtatgata accaagacga tcctcagacg 240 gattgccaac aatgccagaggcgatgcagg cagcaggaga gtggcccacg tcagcaacaa 300 tactgccaac gacgctgcaaggaaatatgt gaagaagaag aagaatataa ccgacaacgt 360 gatccacagc agcaatacgagcaatgtcag gagcgctgcc aacggcacga gacagagcca 420 cgtcacatgc aaacatgtcaacaacgctgc gagaggagat atgaaaagga gaaacgtaag 480 caacaaaaga gatatgaagagcaacaacgt gaagacgaag agaaatatga agagcgaatg 540 aaggaagaag ataacaaacgcgatccacaa caaagagagt acgaagactg ccggaggcgc 600 tgcgaacaac aggagccacgtcagcagtac cagtgccagc gaagatgccg agagcagcag 660 aggcaacacg gccgaggtggtgatttgatt aaccctcaga ggggaggcag cggcagatac 720 gaggagggag aagagaagcaaagcgacaac ccctactact tcgacgaacg aagcttaagt 780 acaaggttca ggaccgaggaaggccacatc tcagttctgg agaacttcta tggtagatcc 840 aagcttctac gcgcactaaaaaactatcgc ttggtgctcc tcgaggctaa ccccaacgcc 900 ttcgtgctcc ctacccacttggacgcagat gccattctct tggtcaccgg agggagagga 960 gccctcaaaa tgatccaccgtgacaacaga gaatcctaca acctcgagtg tggagacgta 1020 atcagaatcc cagctggaaccacattctac ttaatcaacc gagacaacaa cgagaggctc 1080 cacatagcca agttcttacagaccatatcc actcctggcc aatacaagga attcttccca 1140 gctggaggcc aaaacccagagccgtacctc agtaccttca gcaaagagat tctcgaggct 1200 gcgctcaaca cacaagcagagaggctgcgt ggggtgcttg gacagcaaag ggagggagtg 1260 ataattagtg cgtcacaggagcagatcagg gagttgactc gagatgactc agagtcacga 1320 cgctggcata taaggagaggtggtgaatca agcaggggac cttacaatct gttcaacaaa 1380 aggccactgt actccaacaaatacggtcaa gcctacgaag tcaaacctga ggactacagg 1440 caactccaag acatggacgtatcggttttc atagccaaca tcacccaggg atccatgatg 1500 ggtcccttct tcaacactaggtctacaaag gtggtagtgg tggctagtgg agaggcagat 1560 gtggaaatgg catgccctcacttgtcggga agacacggcg gccgccgtgg agggaaaagg 1620 catgaggagg aagaggatgtgcactatgag caggttaaag cacgtttgtc gaagagagag 1680 gccattgttg ttccggtaggtcatcccgtc gtcttcgttt catccggaaa cgagaacctg 1740 ctgctttttg catttggaatcaatgcccaa aacaaccacg agaacttcct cgcggggaga 1800 gagaggaacg tgctgcagcagatagagcca caggcaatgg agctagcgtt tgccgctcca 1860 aggaaagagg tagaagagttatttaacagc caggacgagt ctatcttctt tcctgggccc 1920 aggcagcacc agcaacagtcttcccgctcc accaagcaac aacagcctct cgtctccatt 1980 ctggacttcg ttggcttctaaagttctaca aaaaagagtg tgttatgtag tataggttag 2040 tagctcctag ctcggtgtatgcgagtggta agagaccaag acgctaaatc cctaagtaac 2100 taacctggcg agcttgcgtgtatgcaaata aagaggaaca gctttccaac tttaaaaaaa 2160 aaaaaaaaaa a 2171 5 625PRT Macadamia integrifolia PEPTIDE (1)...(625) Partial mature peptide 5Gln Cys Met Gln Leu Glu Thr Ser Gly Gln Met Arg Arg Cys Val Ser 1 5 1015 Gln Cys Asp Lys Arg Phe Glu Glu Asp Ile Asp Trp Ser Lys Tyr Asp 20 2530 Asn Gln Glu Asp Pro Gln Thr Glu Cys Gln Gln Cys Gln Arg Arg Cys 35 4045 Arg Gln Gln Glu Ser Asp Pro Arg Gln Gln Gln Tyr Cys Gln Arg Arg 50 5560 Cys Lys Glu Ile Cys Glu Glu Glu Glu Glu Tyr Asn Arg Gln Arg Asp 65 7075 80 Pro Gln Gln Gln Tyr Glu Gln Cys Gln Lys Arg Cys Gln Arg Arg Glu 8590 95 Thr Glu Pro Arg His Met Gln Ile Cys Gln Gln Arg Cys Glu Arg Arg100 105 110 Tyr Glu Lys Glu Lys Arg Lys Gln Gln Lys Arg Tyr Glu Glu GlnGln 115 120 125 Arg Glu Asp Glu Glu Lys Tyr Glu Glu Arg Met Lys Glu GlyAsp Asn 130 135 140 Lys Arg Asp Pro Gln Gln Arg Glu Tyr Glu Asp Cys ArgArg His Cys 145 150 155 160 Glu Gln Gln Glu Pro Arg Leu Gln Tyr Gln CysGln Arg Arg Cys Gln 165 170 175 Glu Gln Gln Arg Gln His Gly Arg Gly GlyAsp Leu Met Asn Pro Gln 180 185 190 Arg Gly Gly Ser Gly Arg Tyr Glu GluGly Glu Glu Lys Gln Ser Asp 195 200 205 Asn Pro Tyr Tyr Phe Asp Glu ArgSer Leu Ser Thr Arg Phe Arg Thr 210 215 220 Glu Glu Gly His Ile Ser ValLeu Glu Asn Phe Tyr Gly Arg Ser Lys 225 230 235 240 Leu Leu Arg Ala LeuLys Asn Tyr Arg Leu Val Leu Leu Glu Ala Asn 245 250 255 Pro Asn Ala PheVal Leu Pro Thr His Leu Asp Ala Asp Ala Ile Leu 260 265 270 Leu Val IleGly Gly Arg Gly Ala Leu Lys Met Ile His Arg Asp Asn 275 280 285 Arg GluSer Tyr Asn Leu Glu Cys Gly Asp Val Ile Arg Ile Pro Ala 290 295 300 GlyThr Thr Phe Tyr Leu Ile Asn Arg Asp Asn Asn Glu Arg Leu His 305 310 315320 Ile Ala Lys Phe Leu Gln Thr Ile Ser Thr Pro Gly Gln Tyr Lys Glu 325330 335 Phe Phe Pro Ala Gly Gly Gln Asn Pro Glu Pro Tyr Leu Ser Thr Phe340 345 350 Ser Lys Glu Ile Leu Glu Ala Ala Leu Asn Thr Gln Thr Glu ArgLeu 355 360 365 Arg Gly Val Leu Gly Gln Gln Arg Glu Gly Val Ile Ile ArgAla Ser 370 375 380 Gln Glu Gln Ile Arg Glu Leu Thr Arg Asp Asp Ser GluSer Arg Arg 385 390 395 400 Trp His Ile Arg Arg Gly Gly Glu Ser Ser ArgGly Pro Tyr Asn Leu 405 410 415 Phe Asn Lys Arg Pro Leu Tyr Ser Asn LysTyr Gly Gln Ala Tyr Glu 420 425 430 Val Lys Pro Glu Asp Tyr Arg Gln LeuGln Asp Met Asp Val Ser Val 435 440 445 Phe Ile Ala Asn Ile Thr Gln GlySer Met Met Gly Pro Phe Phe Asn 450 455 460 Thr Arg Ser Thr Lys Val ValVal Val Ala Ser Gly Glu Ala Asp Val 465 470 475 480 Glu Met Ala Cys ProHis Leu Ser Gly Arg His Gly Gly Arg Gly Gly 485 490 495 Gly Lys Arg HisGlu Glu Glu Glu Glu Val His Tyr Glu Gln Val Arg 500 505 510 Ala Arg LeuSer Lys Arg Glu Ala Ile Val Val Leu Ala Gly His Pro 515 520 525 Val ValPhe Val Ser Ser Gly Asn Glu Asn Leu Leu Leu Phe Ala Phe 530 535 540 GlyIle Asn Ala Gln Asn Asn His Glu Asn Phe Leu Ala Gly Arg Glu 545 550 555560 Arg Asn Val Leu Gln Gln Ile Glu Pro Gln Ala Met Glu Leu Ala Phe 565570 575 Ala Ala Ser Arg Lys Glu Val Glu Glu Leu Phe Asn Ser Gln Asp Glu580 585 590 Ser Ile Phe Phe Pro Gly Pro Arg Gln His Gln Gln Gln Ser ProArg 595 600 605 Ser Thr Lys Gln Gln Gln Pro Leu Val Ser Ile Leu Asp PheVal Gly 610 615 620 Phe 625 6 2140 DNA Macadamia integrifoliamat_peptide (1)...(1875) partial mature peptide 6 caatgcatgc agttagagacatcaggccag atgcgtcggt gtgtgagtca gtgcgataag 60 agatttgaag aggatatagattggtctaag tatgataacc aagaggatcc tcagacggaa 120 tgccaacaat gccagaggcgatgcaggcag caggagagtg acccacgtca gcaacaatac 180 tgccaacgac gctgcaaggaaatatgtgaa gaagaagaag aatataaccg acaacgtgat 240 ccacagcagc aatacgagcaatgtcagaag cgctgccaac ggcgcgagac agagccacgt 300 cacatgcaaa tatgtcaacaacgctgcgag aggagatatg aaaaggagaa acgtaagcaa 360 caaaagagat atgaagagcaacaacgtgaa gacgaagaga aatatgaaga gcgaatgaag 420 gaaggagata acaaacgcgatccacaacaa agagagtacg aagactgccg gcggcactgc 480 gaacaacagg agccacgtctgcagtaccag tgccagcgaa gatgccaaga gcagcagagg 540 caacacggcc gaggtggcgatttgatgaac cctcagaggg gaggcagcgg cagatacgag 600 gagggagaag agaagcaaagcgacaacccc tactacttcg acgaacgaag cttaagtaca 660 aggttcagga ccgaggaaggccacatctca gttctggaga acttctatgg tagatccaag 720 cttctacgcg cactaaaaaactatcgcttg gtgctcctcg aggctaaccc caacgccttc 780 gtgctcccta cccacttggatgcagatgcc attctcttgg tcatcggagg gagaggagcc 840 ctcaaaatga tccaccgtgacaacagagaa tcctacaacc tcgagtgtgg agacgtaatc 900 agaatcccag ctggaaccacattctactta atcaaccgag acaacaacga gaggctccac 960 atagccaagt tcttacagaccatatccact cctggccaat acaaggaatt cttcccagct 1020 ggaggccaaa acccagagccgtacctcagt accttcagca aagagattct cgaggctgcg 1080 ctcaacacac aaacagagaggctgcgtggg gtgcttggac agcaaaggga gggagtgata 1140 attagggcgt cacaggagcagatcagggag ttgactcgag atgactcaga gtcacgacgc 1200 tggcatataa ggagaggtggtgaatcaagc aggggacctt acaatctgtt caacaaaagg 1260 ccactgtact ccaacaaatacggtcaagcc tacgaagtca aacctgagga ctacaggcaa 1320 ctccaagaca tggacgtatcagttttcata gccaacatca cccagggatc catgatgggt 1380 cccttcttca acactaggtctacaaaggtg gtagtggtgg ctagtggaga ggcagatgtg 1440 gaaatggcat gccctcacttgtcgggaaga cacggcggcc gcggtggagg gaaaaggcat 1500 gaggaggaag aggaggtgcactatgagcag gttagagcac gtttgtcgaa gagagaggcc 1560 attgttgttc tggcaggtcatcccgtcgtc ttcgtttcat ccggaaacga aaacctgctg 1620 ctttttgcat ttggaatcaatgcccaaaac aaccacgaga acttcctcgc ggggagagag 1680 aggaacgtgc tgcagcagatagagccacag gcaatggagc tagcgtttgc cgcttcaagg 1740 aaagaggtag aagagttatttaacagccag gacgagtcta tcttctttcc tgggcccagg 1800 cagcaccagc aacagtcgccccgctccacc aagcaacaac agcctctcgt ctccattctg 1860 gacttcgttg gcttctaaagttctacaaaa aagagtgtgt tatgtagtat aggttagtag 1920 ctcctagctc ggtgtatgagagtggtaaga gactaagacg ctaaatccct aagtaactaa 1980 cctggcgagc ttgcgtgtatgcaaataaag aggaacagct ttccaacttt agaaagctct 2040 tttttttttt ttttttctttctttttctta agaaataaac gaacgtagat tgcggctcaa 2100 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2140 7 525 PRT Theobroma cacao 7 Met Val Ile SerLys Ser Pro Phe Ile Val Leu Ile Phe Ser Leu Leu 1 5 10 15 Leu Ser PheAla Leu Leu Cys Ser Gly Val Ser Ala Tyr Gly Arg Lys 20 25 30 Gln Tyr GluArg Asp Pro Arg Gln Gln Tyr Glu Gln Cys Gln Arg Arg 35 40 45 Cys Glu SerGlu Ala Thr Glu Glu Arg Glu Gln Glu Gln Cys Glu Gln 50 55 60 Arg Cys GluArg Glu Tyr Lys Glu Gln Gln Arg Gln Gln Glu Glu Glu 65 70 75 80 Leu GlnArg Gln Tyr Gln Gln Cys Gln Gly Arg Cys Gln Glu Gln Gln 85 90 95 Gln GlyGln Arg Glu Gln Gln Gln Cys Gln Arg Lys Cys Trp Glu Gln 100 105 110 TyrLys Glu Gln Glu Arg Gly Glu His Glu Asn Tyr His Asn His Lys 115 120 125Lys Asn Arg Ser Glu Glu Glu Glu Gly Gln Gln Arg Asn Asn Pro Tyr 130 135140 Tyr Phe Pro Lys Arg Arg Ser Phe Gln Thr Arg Phe Arg Asp Glu Glu 145150 155 160 Gly Asn Phe Lys Ile Leu Gln Arg Phe Ala Glu Asn Ser Pro ProLeu 165 170 175 Lys Gly Ile Asn Asp Tyr Arg Leu Ala Met Phe Glu Ala AsnPro Asn 180 185 190 Thr Phe Ile Leu Pro His His Cys Asp Ala Glu Ala IleTyr Phe Val 195 200 205 Thr Asn Gly Lys Gly Thr Ile Thr Phe Val Thr HisGlu Asn Lys Glu 210 215 220 Ser Tyr Asn Val Gln Arg Gly Thr Val Val SerVal Pro Ala Gly Ser 225 230 235 240 Thr Val Tyr Val Val Ser Gln Asp AsnGln Glu Lys Leu Thr Ile Ala 245 250 255 Val Leu Ala Leu Pro Val Asn SerPro Gly Lys Tyr Glu Leu Phe Phe 260 265 270 Pro Ala Gly Asn Asn Lys ProGlu Ser Tyr Tyr Gly Ala Phe Ser Tyr 275 280 285 Glu Val Leu Glu Thr ValPhe Asn Thr Gln Arg Glu Lys Leu Glu Glu 290 295 300 Ile Leu Glu Glu GlnArg Gly Gln Lys Arg Gln Gln Gly Gln Gln Gly 305 310 315 320 Met Phe ArgLys Ala Lys Pro Glu Gln Ile Arg Ala Ile Ser Gln Gln 325 330 335 Ala ThrSer Pro Arg His Arg Gly Gly Glu Arg Leu Ala Ile Asn Leu 340 345 350 LeuSer Gln Ser Pro Val Tyr Ser Asn Gln Asn Gly Arg Phe Phe Glu 355 360 365Ala Cys Pro Glu Asp Phe Ser Gln Phe Gln Asn Met Asp Val Ala Val 370 375380 Ser Ala Phe Lys Leu Asn Gln Gly Ala Ile Phe Val Pro His Tyr Asn 385390 395 400 Ser Lys Ala Thr Phe Val Val Phe Val Thr Asp Gly Tyr Gly TyrAla 405 410 415 Gln Met Ala Cys Pro His Leu Ser Arg Gln Ser Gln Gly SerGln Ser 420 425 430 Gly Arg Gln Asp Arg Arg Glu Gln Glu Glu Glu Ser GluGlu Glu Thr 435 440 445 Phe Gly Glu Phe Gln Gln Val Lys Ala Pro Leu SerPro Gly Asp Val 450 455 460 Phe Val Ala Pro Ala Gly His Ala Val Thr PhePhe Ala Ser Lys Asp 465 470 475 480 Gln Pro Leu Asn Ala Val Ala Phe GlyLeu Asn Ala Gln Asn Asn Gln 485 490 495 Arg Ile Phe Leu Ala Gly Arg ProPhe Phe Leu Asn His Lys Gln Asn 500 505 510 Thr Asn Val Ile Lys Phe ThrVal Lys Ala Ser Ala Tyr 515 520 525 8 590 PRT Gossypium hirsutum(cotton) 8 Met Val Arg Asn Lys Ser Ala Cys Val Val Leu Leu Phe Ser LeuPhe 1 5 10 15 Leu Ser Phe Gly Leu Leu Cys Ser Ala Lys Asp Phe Pro GlyArg Arg 20 25 30 Gly Asp Asp Asp Pro Pro Lys Arg Tyr Glu Asp Cys Arg ArgArg Cys 35 40 45 Glu Trp Asp Thr Arg Gly Gln Lys Glu Gln Gln Gln Cys GluGlu Ser 50 55 60 Cys Lys Ser Gln Tyr Gly Glu Lys Asp Gln Gln Gln Arg HisArg Pro 65 70 75 80 Glu Asp Pro Gln Arg Arg Tyr Glu Glu Cys Gln Gln GluCys Arg Gln 85 90 95 Gln Glu Glu Arg Gln Gln Pro Gln Cys Gln Gln Arg CysLeu Lys Arg 100 105 110 Phe Glu Gln Glu Gln Gln Gln Ser Gln Arg Gln PheGln Glu Cys Gln 115 120 125 Gln His Cys His Gln Gln Glu Gln Arg Pro GluLys Lys Gln Gln Cys 130 135 140 Val Arg Glu Cys Arg Glu Lys Tyr Gln GluAsn Pro Trp Arg Gly Glu 145 150 155 160 Arg Glu Glu Glu Ala Glu Glu GluGlu Thr Glu Glu Gly Glu Gln Glu 165 170 175 Gln Ser His Asn Pro Phe HisPhe His Arg Arg Ser Phe Gln Ser Arg 180 185 190 Phe Arg Glu Glu His GlyAsn Phe Arg Val Leu Gln Arg Phe Ala Ser 195 200 205 Arg His Pro Ile LeuArg Gly Ile Asn Glu Phe Arg Leu Ser Ile Leu 210 215 220 Glu Ala Asn ProAsn Thr Phe Val Leu Pro His His Cys Asp Ala Glu 225 230 235 240 Lys IleTyr Leu Val Thr Asn Gly Arg Gly Thr Leu Thr Phe Leu Thr 245 250 255 HisGlu Asn Lys Glu Ser Tyr Asn Ile Val Pro Gly Val Val Val Lys 260 265 270Val Pro Ala Gly Ser Thr Val Tyr Leu Ala Asn Gln Asp Asn Lys Glu 275 280285 Lys Leu Ile Ile Ala Val Leu His Arg Pro Val Asn Asn Pro Gly Gln 290295 300 Phe Glu Glu Phe Phe Pro Ala Gly Ser Gln Arg Pro Gln Ser Tyr Leu305 310 315 320 Arg Ala Phe Ser Arg Glu Ile Leu Glu Pro Ala Phe Asn ThrArg Ser 325 330 335 Glu Gln Leu Asp Glu Leu Phe Gly Gly Arg Gln Ser ArgArg Arg Gln 340 345 350 Gln Gly Gln Gly Met Phe Arg Lys Ala Ser Gln GluGln Ile Arg Ala 355 360 365 Leu Ser Gln Glu Ala Thr Ser Pro Arg Glu LysSer Gly Glu Arg Phe 370 375 380 Ala Phe Asn Leu Leu Ser Gln Thr Pro ArgTyr Ser Asn Gln Asn Gly 385 390 395 400 Arg Phe Phe Glu Ala Cys Pro ProGlu Phe Arg Gln Leu Arg Asp Ile 405 410 415 Asn Val Thr Val Ser Ala LeuGln Leu Asn Gln Gly Ser Ile Phe Val 420 425 430 Pro His Tyr Asn Ser LysAla Thr Phe Val Ile Leu Val Thr Glu Gly 435 440 445 Asn Gly Tyr Ala GluMet Val Ser Pro His Leu Pro Arg Gln Ser Ser 450 455 460 Tyr Glu Glu GluGlu Glu Glu Asp Glu Glu Glu Glu Gln Glu Gln Glu 465 470 475 480 Glu GluArg Arg Ser Gly Gln Tyr Arg Lys Ile Arg Ser Arg Leu Ser 485 490 495 ArgGly Asp Ile Phe Val Val Pro Ala Asn Phe Pro Val Thr Phe Val 500 505 510Ala Ser Gln Asn Gln Asn Leu Arg Met Thr Gly Phe Gly Leu Tyr Asn 515 520525 Gln Asn Ile Asn Pro Asp His Asn Gln Arg Ile Phe Val Ala Gly Lys 530535 540 Ile Asn His Val Arg Gln Trp Asp Ser Gln Ala Lys Glu Leu Ala Phe545 550 555 560 Gly Val Ser Ser Arg Leu Val Asp Glu Ile Phe Asn Ser AsnPro Gln 565 570 575 Glu Ser Tyr Phe Val Ser Arg Gln Arg Gln Arg Ala SerGlu 580 585 590 9 22 PRT Artificial Sequence Peptide 1 from M.integrifolia MiAMP2c in which Cys is replaced with Ala and Tyr isreplaced with Ala, MiAMP2cpep1. 9 Arg Gln Arg Asp Pro Gln Gln Gln AlaGlu Gln Ala Gln Lys Arg Ala 1 5 10 15 Gln Arg Arg Glu Thr Glu 20 10 25PRT Artificial Sequence Peptide 2 from M. integrifolia MiAMP2c,MiAMPcpep2. 10 Pro Arg His Met Gln Ile Ala Gln Gln Arg Ala Glu Arg ArgAla Glu 1 5 10 15 Lys Glu Lys Arg Lys Gln Gln Lys Arg 20 25 11 36 PRTArtificial Sequence Synthetic DNA sequence coding for a leader peptide.11 Ser Glu Gln Ile Asp Asn Met Ala Trp Phe His Val Ser Val Cys Asn 1 510 15 Ala Val Phe Val Val Ile Ile Ile Ile Met Leu Leu Met Phe Val Pro 2025 30 Val Val Arg Gly 35 12 20 DNA Artificial Sequence Primer JPM17which binds to M. integrifolia MiAMP2c. 12 cagcagcagt atgagcagtg 20 1321 DNA Artificial Sequence Primer JMP20, a degenerate primer that bindsto MiAMP2-like sequences. 13 tttttcgtak ckkckttcgc a 21 14 24 DNAArtificial Sequence Primer JPM31 corresponding to the 5′ coding regionof MiAMP2c and containing Nde1 and BamH1 sites. 14 acaccatatg cgacaacgtgatcc 24 15 26 DNA Artificial Sequence Primer JPM32 corresponding to the3′ coding region of MiAMP2c and containing Nde1 and BamH1 sites. 15cgttgttttc tctattccta gggttg 26 16 22 PRT Artificial Sequence Peptidecontaining His tag and Factor Xa cleavage site of PET16b vector. 16 MetGly His His His His His His His His His His Ser Ser Gly His 1 5 10 15Ile Glu Gly Arg His Met 20 17 90 DNA Artificial Sequence TcAMP1 forwardoligonucleotide. 17 gggaattcca tatgtatgag cgtgatcctc gacagcaatacgagcaatgc cagaggcgat 60 gcgagtcgga agcgactgaa gaaagggagc 90 18 91 DNAArtificial Sequence TcAMP1 reverse oligonucleotide. 18 gaagcgactgaagaaaggga gcaagagcag tgtgaacaac gctgtgaaag ggagtacaag 60 gagcagcagagacagcaata gggatccaca c 91 19 101 DNA Artificial Sequence TcAMP2 forwardoligonucleotide. 19 gggaattcca tatgcttcaa aggcaatacc agcaatgtcaagggcgttgt caagagcaac 60 aacaggggca gagagagcag cagcagtgcc agagaaaatg c101 20 102 DNA Artificial Sequence TcAMP2 reverse oligonucleotide. 20gtgtggatcc ctagctccta ttttttttgt gattatggta attctcgtgc tcgcctctct 60cttgttcctt atattgctcc cagcattttc tctggcactg ct 102 21 614 PRT Peanut 21Met Arg Gly Arg Val Ser Pro Leu Met Leu Leu Leu Gly Ile Leu Val 1 5 1015 Leu Ala Ser Val Ser Ala Thr Gln Ala Lys Ser Pro Tyr Arg Lys Thr 20 2530 Glu Asn Pro Cys Ala Gln Arg Cys Leu Gln Ser Cys Gln Gln Glu Pro 35 4045 Asp Asp Leu Lys Gln Lys Ala Cys Glu Ser Arg Cys Thr Lys Leu Glu 50 5560 Tyr Asp Pro Arg Cys Val Tyr Asp Thr Gly Ala Thr Asn Gln Arg His 65 7075 80 Pro Pro Gly Glu Arg Thr Arg Gly Arg Gln Pro Gly Asp Tyr Asp Asp 8590 95 Asp Arg Arg Gln Pro Arg Arg Glu Glu Gly Gly Arg Trp Gly Pro Ala100 105 110 Glu Pro Arg Glu Arg Glu Arg Glu Glu Asp Trp Arg Gln Pro ArgGlu 115 120 125 Asp Trp Arg Arg Pro Ser His Gln Gln Pro Arg Lys Ile ArgPro Glu 130 135 140 Gly Arg Glu Gly Glu Gln Glu Trp Gly Thr Pro Gly SerGlu Val Arg 145 150 155 160 Glu Glu Thr Ser Arg Asn Asn Pro Phe Tyr PhePro Ser Arg Arg Phe 165 170 175 Ser Thr Arg Tyr Gly Asn Gln Asn Gly ArgIle Arg Val Leu Gln Arg 180 185 190 Phe Asp Gln Arg Ser Lys Gln Phe GlnAsn Leu Gln Asn His Arg Ile 195 200 205 Val Gln Ile Glu Ala Arg Pro AsnThr Leu Val Leu Pro Lys His Ala 210 215 220 Asp Ala Asp Asn Ile Leu ValIle Gln Gln Gly Gln Ala Thr Val Thr 225 230 235 240 Val Ala Asn Gly AsnAsn Arg Lys Ser Phe Asn Leu Asp Glu Gly His 245 250 255 Ala Leu Arg IlePro Ser Gly Phe Ile Ser Tyr Ile Leu Asn Arg His 260 265 270 Asp Asn GlnAsn Leu Arg Val Ala Lys Ile Ser Met Pro Val Asn Thr 275 280 285 Pro GlyGln Phe Glu Asp Phe Phe Pro Ala Ser Ser Arg Asp Gln Ser 290 295 300 SerTyr Leu Gln Gly Phe Ser Arg Asn Thr Leu Glu Ala Ala Phe Asn 305 310 315320 Ala Glu Phe Asn Glu Ile Arg Arg Val Leu Leu Glu Glu Asn Ala Gly 325330 335 Gly Glu Gln Glu Glu Arg Gly Gln Arg Arg Arg Ser Thr Arg Ser Ser340 345 350 Asp Asn Glu Gly Val Ile Val Lys Val Ser Lys Glu His Val GlnGlu 355 360 365 Leu Thr Lys His Ala Lys Ser Val Ser Lys Lys Gly Ser GluGlu Glu 370 375 380 Asp Ile Thr Asn Pro Ile Asn Leu Arg Asp Gly Glu ProAsp Leu Ser 385 390 395 400 Asn Asn Phe Gly Arg Leu Phe Glu Val Lys ProAsp Lys Lys Asn Pro 405 410 415 Gln Leu Gln Asp Leu Asp Met Met Leu ThrCys Val Glu Ile Lys Glu 420 425 430 Gly Ala Leu Met Leu Pro His Phe AsnSer Lys Ala Met Val Ile Val 435 440 445 Val Val Asn Lys Gly Thr Gly AsnLeu Glu Leu Val Ala Val Arg Lys 450 455 460 Glu Gln Gln Gln Arg Gly ArgArg Glu Gln Glu Trp Glu Glu Glu Glu 465 470 475 480 Glu Asp Glu Glu GluGlu Gly Ser Asn Arg Glu Val Arg Arg Tyr Thr 485 490 495 Ala Arg Leu LysGlu Gly Asp Val Phe Ile Met Pro Ala Ala His Pro 500 505 510 Val Ala IleAsn Ala Ser Ser Glu Leu His Leu Leu Gly Phe Gly Ile 515 520 525 Asn AlaGlu Asn Asn His Arg Ile Phe Leu Ala Gly Asp Lys Asp Asn 530 535 540 ValIle Asp Gln Ile Glu Lys Gln Ala Lys Asp Leu Ala Phe Pro Gly 545 550 555560 Ser Gly Glu Gln Val Glu Lys Leu Ile Lys Asn Gln Arg Glu Ser His 565570 575 Phe Val Ser Ala Arg Pro Gln Ser Gln Ser Pro Ser Ser Pro Glu Lys580 585 590 Glu Asp Gln Glu Glu Glu Asn Gln Gly Gly Lys Gly Pro Leu LeuSer 595 600 605 Ile Leu Lys Ala Phe Asn 610 22 582 PRT Maize 22 Met ValSer Ala Arg Ile Val Val Leu Leu Ala Thr Leu Leu Cys Ala 1 5 10 15 AlaAla Ala Val Ala Ser Ser Trp Glu Asp Asp Asn His His His His 20 25 30 GlyGly His Lys Ser Gly Gln Cys Val Arg Arg Cys Glu Asp Arg Pro 35 40 45 TrpHis Gln Arg Pro Arg Cys Leu Glu Gln Cys Arg Glu Glu Glu Arg 50 55 60 GluLys Arg Gln Glu Arg Ser Arg His Glu Ala Asp Asp Arg Ser Gly 65 70 75 80Glu Gly Ser Ser Glu Asp Glu Arg Glu Gln Glu Lys Glu Lys Gln Lys 85 90 95Asp Arg Arg Pro Tyr Val Phe Asp Arg Arg Ser Phe Arg Arg Val Val 100 105110 Arg Ser Glu Gln Gly Ser Leu Arg Val Leu Arg Pro Phe Asp Glu Val 115120 125 Ser Arg Leu Leu Arg Gly Ile Arg Asp Tyr Arg Val Ala Val Leu Glu130 135 140 Ala Asn Pro Arg Ser Phe Val Val Pro Ser His Thr Asp Ala HisCys 145 150 155 160 Ile Cys Tyr Val Ala Glu Gly Glu Gly Val Val Thr ThrIle Glu Asn 165 170 175 Gly Glu Arg Arg Ser Tyr Thr Ile Lys Gln Gly HisVal Phe Val Ala 180 185 190 Pro Ala Gly Ala Val Thr Tyr Leu Ala Asn ThrAsp Gly Arg Lys Lys 195 200 205 Leu Val Ile Thr Lys Ile Leu His Thr IleSer Val Pro Gly Glu Phe 210 215 220 Gln Phe Phe Phe Gly Pro Gly Gly ArgAsn Pro Glu Ser Phe Leu Ser 225 230 235 240 Ser Phe Ser Lys Ser Ile GlnArg Ala Ala Tyr Lys Thr Ser Ser Asp 245 250 255 Arg Leu Glu Arg Leu PheGly Arg His Gly Gln Asp Lys Gly Ile Ile 260 265 270 Val Arg Ala Thr GluGlu Gln Thr Arg Glu Leu Arg Arg His Ala Ser 275 280 285 Glu Gly Gly HisGly Pro His Trp Pro Leu Pro Pro Phe Gly Glu Ser 290 295 300 Arg Gly ProTyr Ser Leu Leu Asp Gln Arg Pro Ser Ile Ala Asn Gln 305 310 315 320 HisGly Gln Leu Tyr Glu Ala Asp Ala Arg Ser Phe His Asp Leu Ala 325 330 335Glu His Asp Val Ser Val Ser Phe Ala Asn Ile Thr Ala Gly Ser Met 340 345350 Ser Ala Pro Leu Phe Asn Thr Arg Ser Phe Lys Ile Ala Tyr Val Pro 355360 365 Asn Gly Lys Gly Tyr Ala Glu Ile Val Cys Pro His Arg Gln Ser Gln370 375 380 Gly Gly Glu Ser Glu Arg Glu Arg Asp Lys Gly Arg Arg Ser GluGlu 385 390 395 400 Glu Glu Glu Glu Ser Ser Glu Glu Gln Glu Glu Ala GlyGln Gly Tyr 405 410 415 His Thr Ile Arg Ala Arg Leu Ser Pro Gly Thr AlaPhe Val Val Pro 420 425 430 Ala Gly His Pro Phe Val Ala Val Ala Ser ArgAsp Ser Asn Leu Gln 435 440 445 Ile Val Cys Phe Glu Val His Ala Asp ArgAsn Glu Lys Val Phe Leu 450 455 460 Ala Gly Ala Asp Asn Val Leu Gln LysLeu Asp Arg Val Ala Lys Ala 465 470 475 480 Leu Ser Phe Ala Ser Lys AlaGlu Glu Val Asp Glu Val Leu Gly Ser 485 490 495 Arg Arg Glu Lys Gly PheLeu Pro Gly Pro Glu Glu Ser Gly Gly His 500 505 510 Glu Glu Arg Glu GlnGlu Glu Glu Glu Arg Glu Glu Arg His Gly Gly 515 520 525 Arg Gly Glu ArgGlu Arg His Gly Arg Glu Glu Arg Glu Lys Glu Glu 530 535 540 Glu Arg GluGly Arg His Gly Gly Arg Glu Glu Arg Glu Glu Glu Glu 545 550 555 560 ArgHis Gly Arg Gly Arg Arg Glu Glu Val Ala Glu Thr Leu Met Arg 565 570 575Met Val Thr Ala Arg Met 580 23 33 PRT Maize 23 Arg Ser Gly Arg Gly GluCys Arg Arg Gln Cys Leu Arg Arg His Glu 1 5 10 15 Gly Gln Pro Trp GluThr Gln Glu Cys Met Arg Arg Cys Arg Arg Arg 20 25 30 Gly 24 637 PRTBarley 24 Met Ala Thr Arg Ala Lys Ala Thr Ile Pro Leu Leu Phe Leu LeuGly 1 5 10 15 Thr Ser Leu Leu Phe Ala Ala Ala Val Ser Ala Ser His AspAsp Glu 20 25 30 Asp Asp Arg Arg Gly Gly His Ser Leu Gln Gln Cys Val GlnArg Cys 35 40 45 Arg Gln Glu Arg Pro Arg Tyr Ser His Ala Arg Cys Val GlnGlu Cys 50 55 60 Arg Asp Asp Gln Gln Gln His Gly Arg His Glu Gln Glu GluGlu Gln 65 70 75 80 Gly Arg Gly Arg Gly Trp His Gly Glu Gly Glu Arg GluGlu Glu His 85 90 95 Gly Arg Gly Arg Gly Arg His Gly Glu Gly Glu Arg GluGlu Glu His 100 105 110 Gly Arg Gly Arg Gly Arg His Gly Glu Gly Glu ArgGlu Glu Glu Arg 115 120 125 Gly Arg Gly His Gly Arg His Gly Glu Gly GluArg Glu Glu Glu Arg 130 135 140 Gly Arg Gly Arg Gly Arg His Gly Glu GlyGlu Arg Glu Glu Glu Glu 145 150 155 160 Gly Arg Gly Arg Gly Arg Arg GlyGlu Gly Glu Arg Asp Glu Glu Gln 165 170 175 Gly Asp Ser Arg Arg Pro TyrVal Phe Gly Pro Arg Ser Phe Arg Arg 180 185 190 Ile Ile Gln Ser Asp HisGly Phe Val Arg Ala Leu Arg Pro Phe Asp 195 200 205 Gln Val Ser Arg LeuLeu Arg Gly Ile Arg Asp Tyr Arg Val Ala Ile 210 215 220 Met Glu Val AsnPro Arg Ala Phe Val Val Pro Gly Phe Thr Asp Ala 225 230 235 240 Asp GlyVal Gly Tyr Val Ala Gln Gly Glu Gly Val Leu Thr Val Ile 245 250 255 GluAsn Gly Glu Lys Arg Ser Tyr Thr Val Lys Glu Gly Asp Val Ile 260 265 270Val Ala Pro Ala Gly Ser Ile Met His Leu Ala Asn Thr Asp Gly Arg 275 280285 Arg Lys Leu Val Ile Ala Lys Ile Leu His Thr Ile Ser Val Pro Gly 290295 300 Lys Phe Gln Phe Leu Ser Val Lys Pro Leu Leu Ala Ser Leu Ser Lys305 310 315 320 Arg Val Leu Arg Ala Ala Phe Lys Thr Ser Asp Glu Arg LeuGlu Arg 325 330 335 Leu Phe Asn Gln Arg Gln Gly Gln Glu Lys Thr Arg SerVal Ser Ile 340 345 350 Val Arg Ala Ser Glu Glu Gln Leu Arg Glu Leu ArgArg Glu Ala Ala 355 360 365 Glu Gly Gly Gln Gly His Arg Trp Pro Leu ProPro Phe Arg Gly Asp 370 375 380 Ser Arg Asp Thr Phe Asn Leu Leu Glu GlnArg Pro Lys Ile Ala Asn 385 390 395 400 Arg His Gly Arg Leu Tyr Glu AlaAsp Ala Arg Ser Phe His Ala Leu 405 410 415 Ala Asn Gln Asp Val Arg ValAla Val Ala Asn Ile Thr Pro Gly Ser 420 425 430 Met Thr Ala Pro Tyr LeuAsn Thr Gln Ser Phe Lys Leu Ala Val Val 435 440 445 Leu Glu Gly Glu GlyGlu Val Gln Ile Val Cys Pro His Leu Gly Arg 450 455 460 Glu Ser Glu SerGlu Arg Glu His Gly Lys Gly Arg Arg Arg Glu Glu 465 470 475 480 Glu GluAsp Asp Gln Arg Gln Gln Arg Arg Arg Gly Ser Glu Ser Glu 485 490 495 SerGlu Glu Glu Glu Glu Gln Gln Arg Tyr Glu Thr Val Arg Ala Arg 500 505 510Val Ser Arg Gly Ser Ala Phe Val Val Pro Pro Gly His Pro Val Val 515 520525 Glu Ile Ser Ser Ser Gln Gly Ser Ser Asn Leu Gln Val Val Cys Phe 530535 540 Glu Ile Asn Ala Glu Arg Asn Glu Arg Val Trp Leu Ala Gly Arg Asn545 550 555 560 Asn Val Ile Gly Lys Leu Gly Ser Pro Ala Gln Glu Leu ThrPhe Gly 565 570 575 Arg Pro Ala Arg Glu Val Gln Glu Val Phe Arg Ala GlnAsp Gln Asp 580 585 590 Glu Gly Phe Val Ala Gly Pro Glu Gln Gln Ser ArgGlu Gln Glu Gln 595 600 605 Glu Gln Glu Arg His Arg Arg Arg Gly Asp ArgGly Arg Gly Asp Glu 610 615 620 Ala Val Glu Thr Phe Leu Arg Met Ala ThrGly Ala Ile 625 630 635 25 605 PRT Soybean (Glycine max) 25 Met Met ArgAla Arg Phe Pro Leu Leu Leu Leu Gly Leu Val Phe Leu 1 5 10 15 Ala SerVal Ser Val Ser Phe Gly Ile Ala Tyr Trp Glu Lys Glu Asn 20 25 30 Pro LysHis Asn Lys Cys Leu Gln Ser Cys Asn Ser Glu Arg Asp Ser 35 40 45 Tyr ArgAsn Gln Ala Cys His Ala Arg Cys Asn Leu Leu Lys Val Glu 50 55 60 Lys GluGlu Cys Glu Glu Gly Glu Ile Pro Arg Pro Arg Pro Arg Pro 65 70 75 80 GlnHis Pro Glu Arg Glu Pro Gln Gln Pro Gly Glu Lys Glu Glu Asp 85 90 95 GluAsp Glu Gln Pro Arg Pro Ile Pro Phe Pro Arg Pro Gln Pro Arg 100 105 110Gln Glu Glu Glu His Glu Gln Arg Glu Glu Gln Glu Trp Pro Arg Lys 115 120125 Glu Glu Lys Arg Gly Glu Lys Gly Ser Glu Glu Glu Asp Glu Asp Glu 130135 140 Asp Glu Glu Gln Asp Glu Arg Gln Phe Pro Phe Pro Arg Pro Pro His145 150 155 160 Gln Lys Glu Glu Arg Asn Glu Glu Glu Asp Glu Asp Glu GluGln Gln 165 170 175 Arg Glu Ser Glu Glu Ser Glu Asp Ser Glu Leu Arg ArgHis Lys Asn 180 185 190 Lys Asn Pro Phe Leu Phe Gly Ser Asn Arg Phe GluThr Leu Phe Lys 195 200 205 Asn Gln Tyr Gly Arg Ile Arg Val Leu Gln ArgPhe Asn Gln Arg Ser 210 215 220 Pro Gln Leu Gln Asn Leu Arg Asp Tyr ArgIle Leu Glu Phe Asn Ser 225 230 235 240 Lys Pro Asn Thr Leu Leu Leu ProAsn His Ala Asp Ala Asp Tyr Leu 245 250 255 Ile Val Ile Leu Asn Gly ThrAla Ile Leu Ser Leu Val Asn Asn Asp 260 265 270 Asp Arg Asp Ser Tyr ArgLeu Gln Ser Gly Asp Ala Leu Arg Val Pro 275 280 285 Ser Gly Thr Thr TyrTyr Val Val Asn Pro Asp Asn Asn Glu Asn Leu 290 295 300 Arg Leu Ile ThrLeu Ala Ile Pro Val Asn Lys Pro Gly Arg Phe Glu 305 310 315 320 Ser PhePhe Leu Ser Ser Thr Glu Ala Gln Gln Ser Tyr Leu Gln Gly 325 330 335 PheSer Arg Asn Ile Leu Glu Ala Ser Tyr Asp Thr Lys Phe Glu Glu 340 345 350Ile Asn Lys Val Leu Phe Ser Arg Glu Glu Gly Gln Gln Gln Gly Glu 355 360365 Gln Arg Leu Gln Glu Ser Val Ile Val Glu Ile Ser Lys Glu Gln Ile 370375 380 Arg Ala Leu Ser Lys Arg Ala Lys Ser Ser Ser Arg Lys Thr Ile Ser385 390 395 400 Ser Glu Asp Lys Pro Phe Asn Leu Arg Ser Arg Asp Pro IleTyr Ser 405 410 415 Asn Lys Leu Gly Lys Phe Phe Glu Ile Thr Pro Glu LysAsn Pro Gln 420 425 430 Leu Arg Asp Leu Asp Ile Phe Leu Ser Ile Val AspMet Asn Glu Gly 435 440 445 Ala Leu Leu Leu Pro His Phe Asn Ser Lys AlaIle Val Ile Leu Val 450 455 460 Ile Asn Glu Gly Asp Ala Asn Ile Glu LeuVal Gly Leu Lys Glu Gln 465 470 475 480 Gln Gln Glu Gln Gln Gln Glu GluGln Pro Leu Glu Val Arg Lys Tyr 485 490 495 Arg Ala Glu Leu Ser Glu GlnAsp Ile Phe Val Ile Pro Ala Gly Tyr 500 505 510 Pro Val Val Val Asn AlaThr Ser Asn Leu Asn Phe Phe Ala Ile Gly 515 520 525 Ile Asn Ala Glu AsnAsn Gln Arg Asn Phe Leu Ala Gly Ser Gln Asp 530 535 540 Asn Val Ile SerGln Ile Pro Ser Gln Val Gln Glu Leu Ala Phe Pro 545 550 555 560 Gly SerAla Gln Ala Val Glu Lys Leu Leu Lys Asn Gln Arg Glu Ser 565 570 575 TyrPhe Val Asp Ala Gln Pro Lys Lys Lys Glu Glu Gly Asn Lys Gly 580 585 590Arg Lys Gly Pro Leu Ser Ser Ile Leu Arg Ala Phe Tyr 595 600 605 26 23PRT Stenocarpus sinuatus PEPTIDE (1)...(23) Partial MiAMP2c homologouspeptide. 26 Val Lys Glu Asp His Gln Phe Glu Thr Arg Gly Glu Ile Leu GluCys 1 5 10 15 Tyr Arg Leu Cys Gln Gln Gln 20 27 17 PRT Stenocarpussinuatus PEPTIDE (1)...(27) Partial MiAMP2c homologous peptide. 27 GlnLys His Arg Ser Gln Ile Leu Gly Cys Tyr Leu Xaa Cys Gln Gln Leu 1 5 1015 28 28 PRT Stenocarpus sinuatus PEPTIDE (1)...(28) Partial MiAMP2chomologous peptide. 28 Leu Asp Pro Ile Arg Gln Gln Gln Leu Cys Gln MetArg Cys Gln Gln 1 5 10 15 Gln Glu Lys Asp Pro Arg Gln Gln Gln Gln CysLys 20 25 29 368 DNA Artificial Sequence A synthetic nucleotide sequencewhich can be used for the expression and secretion of MiAMP2c,containing the leader sequence from SEQ ID NO11 and SEQ ID NO5. 29aactctagag cggccgcgtc gactattttt acaacaatta ccaacaacaa caaacaacaa 60acaacattac aattactatt tacaattaca ggatccacaa ca atg gct tgg ttc 114 MetAla Trp Phe 1 cac gtt tct gtt tgt aac gct gtt ttc gtt gtt att att attatt atg 162 His Val Ser Val Cys Asn Ala Val Phe Val Val Ile Ile Ile IleMet 5 10 15 20 ctt ctt atg ttc gtt cct gtt gtt aga ggt aga caa aga gatcct caa 210 Leu Leu Met Phe Val Pro Val Val Arg Gly Arg Gln Arg Asp ProGln 25 30 35 caa caa tac gag caa tgt caa aag agg tgt caa agg aga gag actgag 258 Gln Gln Tyr Glu Gln Cys Gln Lys Arg Cys Gln Arg Arg Glu Thr Glu40 45 50 cct aga cac atg caa att tgt cag caa agg tgt gaa agg agg tac gag306 Pro Arg His Met Gln Ile Cys Gln Gln Arg Cys Glu Arg Arg Tyr Glu 5560 65 aag gag aag agg aag caa caa aag agg tgaggatccg tcgacgcggc 353 LysGlu Lys Arg Lys Gln Gln Lys Arg 70 75 cgcagatcta gacaa 368 30 77 PRTArtificial Sequence A synthetic peptide sequence which can be used forthe expression and secretion of MiAMP2c containing the leader sequencefrom SEQ ID NO11 and peptide sequence from SEQ ID NO5. 30 Met Ala TrpPhe His Val Ser Val Cys Asn Ala Val Phe Val Val Ile 1 5 10 15 Ile IleIle Met Leu Leu Met Phe Val Pro Val Val Arg Gly Arg Gln 20 25 30 Arg AspPro Gln Gln Gln Tyr Glu Gln Cys Gln Lys Arg Cys Gln Arg 35 40 45 Arg GluThr Glu Pro Arg His Met Gln Ile Cys Gln Gln Arg Cys Glu 50 55 60 Arg ArgTyr Glu Lys Glu Lys Arg Lys Gln Gln Lys Arg 65 70 75 31 27 PRTArtificial Sequence Consensus sequence for antimicrobial peptideswherein X is any amino acid. 31 Cys Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa XaaXaa Cys 20 25 32 28 PRT Artificial Sequence Consensus sequence forantimicrobial peptides wherein X is any amino acid. 32 Cys Xaa Xaa CysXaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa XaaCys Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 20 25 33 29 PRT Artificial SequenceConsensus sequence for antimicrobial peptides wherein X is any aminoacid. 33 Cys Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 20 25 3427 PRT Artificial Sequence Consensus sequence for antimicrobialpeptides, wherein X is any amino acid and the first and last X arePhenylalanine or Tyrosine. 34 Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa XaaXaa Xaa 20 25 35 28 PRT Artificial Sequence Consensus sequence forantimicrobial peptides wherein X is any amino acid and the first andlast X are phenylalanine or Tyrosine. 35 Xaa Xaa Xaa Cys Xaa Xaa Xaa CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Cys Xaa Xaa XaaCys Xaa Xaa Xaa Xaa 20 25 36 29 PRT Artificial Sequence Consensussequence for antimicrobial peptides wherein X is any amino acid and thefirst and last X are phenylalanine or Tyrosine. 36 Xaa Xaa Xaa Cys XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaCys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 20 25 37 20 PRT Artificial SequenceConsensus sequence for antimicrobial peptides wherein X is any aminoacid. 37 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys1 5 10 15 Xaa Xaa Xaa Cys 20 38 21 PRT Artificial Sequence Consensussequence for antimicrobial peptides wherein X is any amino acid. 38 CysXaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15Cys Xaa Xaa Xaa Cys 20 39 22 PRT Artificial Sequence Consensus sequencefor antimicrobial peptides wherein X is any amino acid. 39 Cys Xaa XaaXaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa CysXaa Xaa Xaa Cys 20 40 5 PRT Artificial Sequence Consensus sequence forantimicrobial peptides wherein X is any amino acid. 40 Cys Xaa Xaa XaaCys 1 5

1. A protein fragment having antimicrobial activity, wherein saidprotein fragment is selected from: (ii) a polypeptide having an aminoacid sequence selected from: residues 29 to 73 of SEQ ID NO: 1 residues74 to 116 of SEQ ID NO: 1 residues 117 to 185 of SEQ ID NO: 1 residues186 to 248 of SEQ ID NO: 1 residues 29 to 73 of SEQ ID NO: 3 residues 74to 1 16 of SEQ ID NO: 3 residues 117 to 185 of SEQ ID NO: 3 residues 186to 248 of SEQ ID NO: 3 residues 1 to 32 of SEQ ID NO: 5 residues 33 to75 of SEQ ID NO: 5 residues 76 to 144 of SEQ ID NO: 5 residues 145 to210 of SEQ ID NO: 5 residues 34 to 80 of SEQ ID NO: 7 residues 81 to 140of SEQ ID NO: 7 residues 33 to 79 of SEQ ID NO: 8 residues 80 to 109 ofSEQ ID NO: 8 residues 120 to 161 of SEQ ID NO: 8 residues 32 to 91 ofSEQ ID NO: 21 residues 25 to 84 of SEQ ID NO: 22 residues 29 to 94 ofSEQ ID NO: 24 residues 31 to 85 of SEQ ID NO: 25 residues 1 to 23 of SEQID NO: 26 residues 1 to 17 of SEQ ID NO: 27 residues 1 to 28 of SEQ IDNO: 28; (ii) a homologue of (i); (iii) a polypeptide containing arelative cysteine spacing of C-2X-C-3X-C-(10-12)X-C-3X-C-3X-C wherein Xis any amino acid residue, and C is cysteine; (iv) a polypeptidecontaining a relative cysteine and tyrosine/phenylalanine spacing ofZ-2X-C-3X-C-(10-12)X-C-3X-C-3X-Z wherein X is any amino acid residue,and C is cysteine, and Z is tyrosine or phenylalanine; (v) a polypeptidecontaining a relative cysteine spacing of C-3X-C-(10-12)X-C-3X-C whereinX is any amino acid residue, and C is cysteine; (vi) a polypeptide withsubstantially the same spacing of positively charged residues relativeto the spacing of cysteine residues as (i); and (vii) a fragment of thepolypeptide of any one of (i) to (vi) which has substantially the sameantimicrobial activity as (i).
 2. A protein containing at least onepolypeptide fragment according to claim 1, wherein said polypeptidefragment has a sequence selected from within a sequence comprising SEQID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5
 3. A protein having a sequenceselected from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:
 5. 4. An isolatedor synthetic DNA encoding a polypeptide fragment according to claim 1.5. The DNA according to claim 4, wherein said DNA has a sequenceselected from SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:
 6. 6. A DNAconstruct which includes a DNA according to claim 4 operatively linkedto elements for the expression of said encoded protein.
 7. A transgenicplant harbouring a DNA construct according to claim
 6. 8. The transgenicplant according to claim 7, wherein said plant is a monocotyledonousplant or a dicotyledonous plant.
 9. The transgenic plant according toclaim 7, wherein said plant is selected from maize, banana, peanut,field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats,potato, soybeans, cotton, carnations, roses, or sorghum. 10.Reproductive material of a transgenic plant according to claim
 7. 11. Acomposition comprising an antimicrobial protein according to claim 1together with an agriculturally-acceptable carrier diluent or excipient.12. A composition comprising an antimicrobial protein according to claim1 together with an pharmaceutically-acceptable carrier diluent orexcipient.
 13. A method of controlling microbial infestation of a plant,the method comprising: i) treating said plant with an antimicrobialprotein according to claim 1 or a composition according to claim 11; orii) introducing a DNA construct according to claim 6 into said plant.14. A method of controlling microbial infestation of a mammalian animal,the method comprising treating the animal with an antimicrobial proteinaccording to claim 1 or a composition according to claim
 12. 15. Themethod of claim 14, wherein said mammalian animal is a human.
 16. Amethod of preparing an antimicrobial protein, which method comprises thesteps of: a) obtaining or designing an amino acid sequence which forms ahelix-turn-helix structure; b) replacing individual residues to achievesubstantially the same distribution of positively charged residues andcysteine residues as in one or more of the amino acid sequences shown inFIG. 4; c) synthesising a protein comprising said amino acid sequencechemically or by recombinant DNA techniques in liquid culture; and d) ifnecessary, forming disulphide linkages between said cysteine residues.